WO2013107388A1

WO2013107388A1 - Interferon and immune globulin fc section fusion protein

Info

Publication number: WO2013107388A1
Application number: PCT/CN2013/070693
Authority: WO
Inventors: 郭村勇; 吴忠晋; 陈汉婷
Original assignee: 施怀哲维克生物科技股份有限公司
Priority date: 2012-01-19
Filing date: 2013-01-18
Publication date: 2013-07-25
Also published as: BR112014017788A2; PH12014501643A1; CN103214579B; CN103214579A; KR20150002588A; CN105001340A

Abstract

Provided is a fusion protein containing an animal interferon and an animal immune globulin Fc section. The fusion protein further comprises a linker for linking the interferon and the immune globulin Fc section. Further provided are a polyribonucleotide for encoding the fusion protein, a method for preparing the fusion protein, and an application of the fusion protein in the preparation of an antiviral drug.

Description

干扰素和免疫球蛋白 FC段的融合蛋白據页域 Fusion protein of interferon and immunoglobulin FC segment

本发明关于一种动物融合重组型干扰素，特别是关于一种具有抗动物病毒活性的融合重组型干扰素。背景财 The present invention relates to an animal fusion recombinant interferon, and more particularly to a fusion recombinant interferon having antiviral activity. Background

干扰素最早是在 1957年由英国学者 Alick Isaacs和 Jean Lindenmann在进行流感病毒试验中所发现，当细顔受病毒感染后，会立即制造一种细胞激素，诱发邻遞田 )1^生抗病毒蛋白，干扰病毒的复制。该细胞激素随后被命名为干扰素 (Interferon, IF )。干扰素的抗病毒作用，主要是由第一型干扰素 (IFN _α/β)所负责。干扰素除了具有抗病毒作用外，还具有抗肿瘤、 \mm 胞分化和免疫调节等功用。 Interferon was first discovered in 1957 by British scholars Alick Isaacs and Jean Lindenmann in the influenza virus test. When the skin is infected by the virus, it will immediately produce a cytokine, which induces the neighboring field. Protein, interferes with the replication of the virus. This cytokine was subsequently named Interferon (IF). The antiviral effect of interferon is mainly responsible for the first type of interferon (IFN _α /β). In addition to its antiviral effect, interferon also has anti-tumor, \mm cell differentiation and immune regulation.

目前市面上的干扰素制剂，多为针对人麵开发设计，例如：用来治疗人类 B型与 C型肝炎等病毒性疾病以及卡波西氏瘤 (Kaposi ' s sarcoma, KS)、黑色素肿瘤 (malignant melanoma)等月中瘤疾病上的干扰素。 Currently, interferon preparations on the market are mostly designed for human face development, for example: for the treatment of viral diseases such as hepatitis B and C, as well as Kaposi's sarcoma (KS), melanoma ( Malignant melanoma) Interferon on tumor diseases in the middle of the month.

至于经济动面，以干扰素治疗病毒性疾病的情形并不普遍。若无有效的疫苗来预防某一动物病毒，当畜群受该病毒感染时，只能使用支持性疗法处理，但¾¾往往不彰，而造成畜牧业重大损失；因此，动物干扰素的开发，对动物 ί纖产业而言，可说是刻不容艱极为重要的事。发明内容 As for economic development, it is not common to treat viral diseases with interferon. If there is no effective vaccine to prevent an animal virus, when the herd is infected with the virus, it can only be treated with supportive therapy, but it is often not recognized, resulting in significant loss of animal husbandry; therefore, the development of animal interferon, For the animal industry, it can be said that it is extremely difficult to be difficult. Summary of the invention

本发明于第一部份中共一种动物融合重组型干扰素。由于干扰素属于小分子蛋白质，在体内半衰期短 (约 2-8小时)且不稳定，因此本发明所共的动物融合重组型干扰素是将动物干扰素蛋白与半衰期较长的动物免疫球蛋白 IgG-Fc片段融合，而形成较稳定的动物融合重组型干扰素。于一较佳实施例中，该动物干扰素蛋白以及该动物免疫球蛋白 IgG-Fc片段是以由憶酸 (Glycine, G) i t (Serine, S) j¾的连接子 (1 inker)戶； ¾接起来的。 In the first part of the invention, an animal fusion recombinant interferon is used. Since interferon belongs to small molecular proteins, it has a short half-life (about 2-8 hours) in vivo and is unstable. Therefore, the animal fusion recombinant interferon of the present invention is an animal interferon protein and an animal immunoglobulin with a long half-life. The IgG-Fc fragment is fused to form a more stable animal fusion recombinant interferon. In a preferred embodiment, the animal interferon protein and the animal immunoglobulin IgG-Fc fragment are a linker (1 inker) of Glycine (G) it (Serine, S) j3⁄4; Pick up.

于一实施例中，该干扰素蛋白为猪干扰素 α，该猪干扰素 α系与如 SEQ ID Nos : 2、 17、 19、 21、 23、 25、 27、 29、 31、 33、 35、 37、 39、 41、 43、 45及 47戶;^之氨基酉辨列其中至少一者具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该猪干扰素 α 选自于由如 SEQ ID Nos : 2、 17、 19、 21、 23、 25、 27、 29、 31、 33、 35、 37、 39、 41、 43、 45 及 47戶； ^之氨基酸序列腺且成之群组中至少一者。 In one embodiment, the interferon protein is porcine interferon alpha, and the porcine interferon alpha is as SEQ ID Nos: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47; wherein at least one of the aminoguanidines has at least 80% sequence homology, preferably, has 85% sequence homology, and more preferably, has 90 % sequence homology, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology. In a preferred embodiment, the porcine interferon alpha is selected from the group consisting of SEQ ID Nos: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47 households; ^ at least one of the amino acid sequence glands and groups.

于一实施例中，该动物免疫球蛋白 IgG-Fc片段为猪免疫球蛋白 IgG-Fc片段，该猪免疫球蛋白 IgG-Fc片段与如 SEQ ID Nos : 4、 49、 51、 53及 55所示之氨基酸序列其中至少一者具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该猪免疫球蛋白 IgG-Fc 片段选自于由如 SEQ ID Nos : 4、 49、 51、 53及 55所示之氨基酸序列所组成之群组中至少一者。 In one embodiment, the animal immunoglobulin IgG-Fc fragment is a porcine immunoglobulin IgG-Fc fragment, and the porcine immunoglobulin IgG-Fc fragment is as set forth in SEQ ID Nos: 4, 49, 51, 53 and 55 At least one of the indicated amino acid sequences has at least 80% sequence homology, preferably 85% sequence homology, more preferably 90% sequence homology, even 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% sequence homology. In a preferred embodiment, the porcine immunoglobulin IgG-Fc fragment is selected from at least one of the group consisting of the amino acid sequences set forth in SEQ ID Nos: 4, 49, 51, 53 and 55.

于一实施例中，该连接子系与如 SEQ ID No: 6所示之 S,列具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该连接子具有如 SEQ ID No： 6所示之 S 酸序列。 In one embodiment, the linker has at least 80% sequence homology to the S, as set forth in SEQ ID No: 6, preferably 85% sequence homology, and more preferably, 90. % sequence homology, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology. In a preferred embodiment, the linker has an S acid sequence as set forth in SEQ ID No: 6.

于一实删中，该动物融合重组型干扰素系与如 SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、 69、 71、 73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、 101、 103、 105、 107、 109、 111、 113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、 143、 145、 147、 149、 151、 153、 155、 157、 159、 161、 163、 165、 167、 169、 1 177、 179、 181、 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、 211、 213、 215、 217、 219、 221及 223戶； ^之氨基酸序列其中之一具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该动物融合重组型干扰素系选自于由如 SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、 69、 71、 73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、 101、 103、 105、 107、 109、 111、 113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、 143、 145、 147、 149、 151、 153、 155、 157、 159、 161、 163、 165、 167、 169、 171、 173、 175、 177、 179、 181、 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、 211、 213、 215、 217、 219、 221及 223所示之 S酉辨列戶獵成之群组。 In an actual deletion, the animal fusion recombinant interferon is as SEQ ID Nos: 8, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 1 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197 , 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 and 223; one of the amino acid sequences has at least 80% sequence homology, preferably 85 % sequence homology, more preferably, 90% sequence homology, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology . In a preferred embodiment, the animal fusion recombinant interferon is selected from the group consisting of SEQ ID Nos: 8, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 , 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 , 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179 , 181, 185, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, and 223 Listed by the households.

本发明于第二部分中共一种编码上述动物融合重组型干扰素的多核苷酸。本发明所^ f共之动物融合重组型干扰素是藉由基因转殖技术而得。首先将编码动物干扰素蛋白的 DNA序列，以及编码动物免疫球蛋白 IgG-Fc片段的 DNA序列选殖到表现载体***中，形成含有编码动物融合重组型干扰素之 DNA序列的质体，翔冬该质#$专殖到表 ***中，经诱导蛋白质表现后而得到动物融合重组型干扰素。 In the second part of the invention, a polynucleotide encoding the above animal fusion recombinant interferon is used. The animal fusion recombinant interferon of the present invention is obtained by gene transfer technology. First, a DNA sequence encoding an animal interferon protein, and a DNA sequence encoding an animal immunoglobulin IgG-Fc fragment are selected into a expression vector system to form a plastid containing a DNA sequence encoding an animal fusion recombinant interferon. The substance #$ is colonized into the surface system, and the animal fusion recombinant interferon is obtained after the protein expression is induced.

于一较佳实施例中，除了将编码动物干扰素蛋白的 DNA序列以及编码动物免疫球蛋白 IgG-Fc 片段的 DNA序列选殖到表现载体***中，并将编码由甘氨¾¾丝氨酸组成的连接子 (1 inker)的 DNA 序列选殖到该表现载体***中，以连接该编码动物干扰素蛋白的 DNA序列以及编码动物免疫球蛋白 IgG-Fc片段的 DNA序列。 In a preferred embodiment, the DNA sequence encoding the animal interferon protein and the DNA sequence encoding the animal immunoglobulin IgG-Fc fragment are selected into the expression vector system and the linkage encoding the glycine 3⁄43⁄4 serine is encoded. A DNA sequence of 1 inker was cloned into the expression vector system to ligate the DNA sequence encoding the animal interferon protein and the DNA sequence encoding the animal immunoglobulin IgG-Fc fragment.

于一实施例中，该编码动物干扰素蛋白的 DNA序列系与如 SEQ ID Nos : U 16、 18、 20、 22、 24、 26、 28、 30、 32、 34、 36、 38、 40、 42、 44及 46所示之 DNA序列其中至少一者具有至少 80% 序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该编码动物干扰素蛋白的 DNA序列系选自于由如 SEQ ID Nos : 1、 16、 18、 20、 22、 24、 26、 28、 30、 32、 34、 36、 38、 40、 42、 44及 46 DNA序列戶形且成之群组中至少一者。 In one embodiment, the DNA sequence encoding the animal interferon protein is as SEQ ID Nos: U 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 At least one of the DNA sequences shown at 44, 46 has at least 80% sequence homology, preferably 85% sequence homology, more preferably 90% sequence homology, or even 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology. In a preferred embodiment, the DNA sequence encoding the animal interferon protein is selected from the group consisting of SEQ ID Nos: 1, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 , 38, 40, 42, 44, and 46 DNA sequence houses and at least one of the groups.

于一实施例中，该编码动物免疫球蛋白 IgG-Fc片段的 DNA序列与如 SEQ ID Nos : 3、 48、 50、 52及 54所示之 DNA序列其中至少一者具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源 I"生，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该编码动物免疫球蛋白 IgG-Fc片段的 DNA序列选自于由如 SEQ ID Nos： 3、 48、 50、 52及 54戶; ^之 DNA序列所賊之群组中至少一者。 In one embodiment, the DNA sequence encoding the animal immunoglobulin IgG-Fc fragment has at least 80% sequence homology with at least one of the DNA sequences set forth in SEQ ID Nos: 3, 48, 50, 52 and 54 Sexually, preferably, has 85% sequence homology, and more preferably, has 90% sequence homology I", even 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% sequence homology. In a preferred embodiment, the DNA sequence encoding the animal immunoglobulin IgG-Fc fragment is selected from the group consisting of SEQ ID Nos: 3, 48, 50, 52 and 54 At least one of the group of thieves of the DNA sequence.

于一实施例中，该编码由甘氨酉氨酸 «的连接子 (linker)的 DNA序列与如 SEQ ID No: 5戶； ^之 DNA序列具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该编码由酉氨酸賊的连接子的 DNA序列系具有如 SEQ ID No: 5麻之 DNA序列。 In one embodiment, the DNA sequence encoding the linker of glycine lysine has at least 80% sequence homology with the DNA sequence of SEQ ID No: 5; preferably, Having 85% sequence homology, more preferably, 90% sequence homology, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence Source. In a preferred embodiment, the DNA sequence encoding the linker of the valine thief has a DNA sequence as SEQ ID No: 5.

于一实施例中，该编码动物融合重组型干扰素的 DNA序列与如 SEQ ID Nos : 7、 56、 58、 60、 62、 64、 66、 68、 70、 72、 74、 76、 78、 80、 82、 84、 86、 88、 90、 92、 94、 96、 98、 100、 102、 104、 106、 108、 110、 112、 114、 116、 118、 120、 122、 124、 126、 128、 130、 132、 134、 136、 138、 140、 142、 144、 146、 148、 150、 152、 154、 156、 158、 160、 162、 164、 166、 168、 170、 172、 174、 176、 178、 180、 182、 184、 186、 188、 190、 192、 194、 196、 198、 200、 202、 204、 206、 208、 210、 212、 214、 216、 218、 220及 222所示之 DNA序列具有至少 80%序列同源性，较佳者，具有 85%序列同源性，更佳者，具有 90%序列同源性，甚至是 91%、 92%、 93%、 94%、 95%、 96%、 97%、 98%、 99%序列同源性。于一较佳实施例中，该编码动物融合重组型干扰素的 DNA序列具有如 SEQ ID Nos : 7、 56、 58、 60、 62、 64、 66、 68、 70、 72、 74、 76、 78、 80、 82、 84、 86、 88、 90、 92、 94、 96、 98、 100、 102、 104、 106、 108、 110、 112、 114、 116、 11 124、 126、 128、 130、 132、 134、 136、 138、 140、 142、 144、 146、 148、 150、 152、 154、 156、 158、 160、 162、 164、 166、 168、 170、 172、 174、 176、 178、 180、 182、 184、 186、 188、 190、 192、 194、 196、 198、 200、 202、 204、 206、 208、 210、 212、 214、 216、 218、 220及 222所示之 DNA序列。 In one embodiment, the DNA sequence encoding the animal fusion recombinant interferon is as SEQ ID Nos: 7, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 , 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 , 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180 The DNA sequences shown at 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, and 222 have at least 80 % sequence homology, preferably, with 85% sequence homology, more preferably, 90% sequence homology, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence homology. In a preferred embodiment, the DNA sequence encoding the animal fusion recombinant interferon has SEQ ID Nos: 7, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 , 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 11 124, 126, 128, 130, 132, 134, 136, 138, 140, 142 , 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192 DNA sequences as shown at 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, and 222.

该表现载体可为原核生物表现载体或纖生物表现载体。该原核生物表现载体包含但不限于 pET系列表现载体以及 pGEX系列表现载体。该真核生物表现载体包含但不限于 pSecTag系列表现载体。 The expression vector can be a prokaryotic expression vector or a fibrinous expression vector. The prokaryotic expression vector includes, but is not limited to, a pET series expression vector and a pGEX series expression vector. The eukaryotic expression vector includes, but is not limited to, the pSecTag series expression vector.

该表现***可为原核生物表 *** (如：细菌)或餓生物表现*** (如：酵母菌、昆虫细胞、植物细胞和哺乳动物细胞等)。于一实施例中，该表 am统为大肠杆菌 (^scAer cA s coli)。于另一实施例中，该表现***为哺乳动物细胞。可用于本发明动物融合重组型干扰素表现的哺乳动物细胞包含但不限于 3T3细胞、中国仓鼠卵巢细胞 (Chinese hamster ovary cells, CHO cells)、幼鼠肾细胞 (baby hamster kidney cells, BHK cells)、人类子***细胞 (HeLa cells) , 以及人概癌细胞 (HepG2 cells)等。 The performance system can be a prokaryotic system (eg, bacteria) or a hungry biological expression system (eg, yeast, insect cells, plant cells, and mammalian cells, etc.). In one embodiment, the table is E. coli (^scAer cA s coli). In another embodiment, the expression system is a mammalian cell. Mammalian cells useful for the expression of the recombinant fusion interferon of the present invention include, but are not limited to, 3T3 cells, Chinese hamster ovary cells (CHO cells), baby hamster kidney cells (BHK cells), Human cervical cancer cells (HeLa cells), and human cancer cells (HepG2 cells).

本发明于第三部分中共一种以哺乳细胞产制本发明的动物融合重组型干扰素的优化制程。将带有编码本发明的动物融合重组型干扰素的多核苷酸的哺乳细胞先以含有血清的培养基培养，待该细赃长稳定后，再改为青培养基培养，于每隔 5天更换新的青培养基，并收取细胞培养基，以获得本发明的动物融合重组型干扰素。 The present invention is directed to an optimized process for producing an animal fusion recombinant interferon of the present invention in a third aspect. A mammalian cell carrying a polynucleotide encoding the animal fusion recombinant interferon of the present invention is first cultured in a serum-containing medium, and after the fine sputum is stably stabilized, it is changed to a sap culture medium every 5 days. The new cyan medium is replaced and the cell culture medium is taken to obtain the animal fusion recombinant interferon of the present invention.

该哺乳动物细胞包含但不限于 3T3细胞、中国仓鼠卵巢细胞 (CHO cells)、幼鼠肾细胞 (BHK cells) , 人类子***细胞 (HeLa cells) , 以及人类肝癌细胞 (HepG2 cells)等。于一实施例中，该哺乳细胞为中国仓鼠卵巢细胞 (CHO cells) The mammalian cells include, but are not limited to, 3T3 cells, Chinese hamster ovary cells (CHO cells), young mouse kidney cells (BHK cells), human cervical cancer cells (HeLa cells), and human liver cancer cells (HepG2 cells). In one embodiment, the mammalian cell is a Chinese hamster ovary cell (CHO cells)

该血清包含但不限于牛血清以及马血清。于一实施例中，该血清为胎牛血清 (fetal bovine serum, FBS)。该血清在培养基中的为 0. 1-10% (v/v)；于一实施例中，该血清为 5% (v/v)。 The serum includes, but is not limited to, bovine serum and horse serum. In one embodiment, the serum is fetal bovine serum (FBS). The serum is 0. 1-10% (v/v) in the medium; in one embodiment, the serum is 5% (v/v).

本发明于第四部分中提供一种动物融合重组型干扰素在制备动物抗病毒药物中的用途。经试验证明，本发明所提供的动物融合重组型干扰素具有抗病毒效果。分别以 DNA病毒 (如：伪狂犬病病毒， pseudorabies virus, PRV)以及 RNA病毒（如：猪繁殖与呼吸道综合症病毒， porcine reproductive and respiratory syndrome virus, PRRSV)为体夕卜试验 (in vitro)测试对象，测试本发明动物融合重组型干扰素的抗病毒活性。结果显示，本发明动物融合重组型干扰素可以有效抑制 DNA病毒以及 R A病毒的宿主细胞内的增殖，且抗病毒效果优于未与动物免疫球蛋白 IgG-Fc 片段融合重组的动物干扰素。因此，本发明所共的动物融合重组型干扰素可用来抑制动物病毒在动物体内增殖。 The present invention provides, in a fourth aspect, the use of an animal fusion recombinant interferon for the preparation of an animal antiviral drug. It has been verified by experiments that the animal fusion recombinant interferon provided by the present invention has an antiviral effect. DNA viruses (eg, pseudorabies virus, PRV) and RNA viruses (eg, porcine reproductive and respiratory syndrome virus, PRRSV) are in vitro test subjects. The antiviral activity of the animal fusion recombinant interferon of the present invention was tested. The results show that the animal fusion recombinant interferon of the present invention can effectively inhibit the proliferation of DNA virus and the host cell of the R A virus, and the antiviral effect is superior to the animal interferon which has not been fused with the animal immunoglobulin IgG-Fc fragment. Therefore, the animal fusion recombinant interferon of the present invention can be used to inhibit the proliferation of animal viruses in animals.

本说明书中腿的所有技术性及科学术语，除非另夕卜有所定义，皆为该所属领域具有通常技艺者可共同了解的意义。 All technical and scientific terms of the legs in this specification, unless otherwise defined, are of common interest to those of ordinary skill in the art.

本发明以下面的实施例予以示范阐明，但本发明不受下述实施例所限制。附图说明 The invention is illustrated by the following examples, but the invention is not limited by the following examples. DRAWINGS

图 1A为带有编码猪融合重组型干扰素基因的质体转染至 CH0细胞后，以 Zeocin抗生素筛选后以 IFA分析的结果；图 1B为 pSecT_ag2 (B)载体转染至 CH0细胞后，以 Zeocin抗生素筛选后以 IFA分析的结果。 Figure 1A shows the results of IFA analysis after screening with Zeocin antibiotics after transfection of plastids encoding the porcine fusion recombinant interferon gene into CH0 cells; Figure 1B shows the transfection of pSecT _a g2 (B) vector into CH0 cells. , the results of IFA analysis after screening with Zeocin antibiotics.

图 2为将带有编码猪融合重组型干扰素基因的质体或将 _PSecTag2 (B)载#$专染至 CH0细胞后，以 Zeocin抗生素筛选后以 ELISA分析的结果。 Fig. 2 shows the results of ELISA analysis after screening with Zeocin antibiotics by plastids encoding the porcine fusion recombinant interferon gene or _P SecTag2 (B)-specific staining to CH0 cells.

图 3为以不同抗体侦测带有编码猪融合重组型干扰素基因的 CH0细胞所表现的蛋白中是否含有猪融合重组型干扰素的结果；第 1道：带有编码猪融合重组型干扰素基因的 CH0细麵表现的蛋白的 SDS-PAGE分析图；第 2道：以 mouse ant i ΙΚ α单株抗体分析；第 3道：以 mouse anti His 单株抗体分析；第 4道：以 goat anti porcine IgG抗体分析。 Figure 3 shows the results of detecting whether the protein expressed by CH0 cells encoding the porcine fusion recombinant interferon gene contains porcine fusion recombinant interferon by different antibodies; lane 1: with coding porcine fusion recombinant interferon SDS-PAGE analysis of the protein expressed by the CH0 fine surface of the gene; lane 2: analysis by mouse ant i ΙΚ α monoclonal antibody; lane 3: by mouse anti His Single antibody analysis; Lane 4: Analysis with goat anti porcine IgG antibody.

图 4为猪融合重组型干扰素 (P IFN-Fc组)及未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFN迎的抗 PRRS病毒及抗 PR病毒的活性比较。具体¾½¾ "式 Figure 4 is a comparison of porcine fusion recombinant interferon (P IFN-Fc group) and interferon (P IFN incubated against PRRS virus and anti-PR virus) that has not been fused with porcine immunoglobulin IgG-Fc fragment. Specific 3⁄41⁄23⁄4 "style

雄例一猪干鐘 αΐ基因选殖 Male case 1 pig trunk clock αΐ gene colonization

取三品系杂交猪 (L X Y-D)的血液分离***血液单核细胞 (Peripheral blood mononuclear cell, PBMC) , 并以异硫氰酉舰 (guanidine thiocyanate, GTC)法萃 ¾ ^、靈 (total靈）。接着将萃取的总 R A进行反转录聚合酶连锁 (reverse polymerase chain reaction, RT-PCR)；先将萃取的 RNA 以 70。C作用 3 中后，将含有 20 μΐ 总靈、 10 μΐ 5χ反应液、 8 μΐ 1. 25mM d TP、 1 μΐ 3' 端互补弓 I子、 11 μΐ蒸馏水、 0. 5 μΐ R asin以及 0. 5 μΐ AMV反转录酶的反应管置于 42。C作用 30 中，以进行 cDNA合成；翔冬合成后的 cDNA进行聚合酶连锁反应 (polymerase chain reaction, PCR)以增殖猪干扰素 al基因 (P IFNal)，在管中力口入 10 μΐ cDNA、 5 μΐ 10x PCR 液、 8 μΐ 1. 25 mM d TP、 1 μΐ 5' 端正向引子、 1 μΐ 3' 端反向弓 I子、 24 μΐ蒸馏水、 1 μΐ Taq聚合酶，混合均匀后方 PCR 反应器中 (Applied Biosystems GeneAmp PCR system 2400)，反应条件为先以 95。C 5 中将 DNA变性，接着以 95°C 1 中、 55°C 30秒、 72°C 30秒进行 30个循环，最后以 72°C 5 中完成 PCR 。其中，猪干扰素 al基因 (P IFNal)的特异性引子序列如下：正向引子（IF -F1) : Peripheral blood mononuclear cells (PBMC) were isolated from the blood of the three-line hybrid pig (L X Y-D) and extracted with guanidine thiocyanate (GTC). The extracted total R A is then subjected to reverse polymerase chain reaction (RT-PCR); the extracted RNA is first 70. After C action 3, it will contain 20 μΐ total, 10 μΐ 5χ reaction solution, 8 μΐ 1. 25mM d TP, 1 μΐ 3′ end complementary bow I, 11 μΐ distilled water, 0.5 μΐ R asin and 0. A reaction tube of 5 μΐ AMV reverse transcriptase was placed at 42. C-acting 30, for cDNA synthesis; the cDNA after Asahi synthesis is subjected to polymerase chain reaction (PCR) to proliferate porcine interferon al gene (P IFNal), and 10 μΐ cDNA is inserted into the tube. 5 μΐ 10x PCR solution, 8 μΐ 1. 25 mM d TP, 1 μΐ 5' forward primer, 1 μΐ 3' end reversed bow I, 24 μΐ distilled water, 1 μΐ Taq polymerase, mixed homogeneous PCR reactor Medium (Applied Biosystems GeneAmp PCR system 2400), the reaction conditions are first 95. The DNA was mutated in C 5 , followed by 30 cycles of 95 ° C 1 , 55 ° C for 30 seconds, 72 ° C for 30 seconds, and finally PCR at 72 ° C 5 . Among them, the specific primer sequence of the porcine interferon al gene (P IFNal) is as follows: Forward primer (IF-F1):

5， -CCCAAGCTTATGGCCCCAACCTCAGCC-3 (SEQ ID NO: 9) 5, -CCCAAGCTTATGGCCCCAACCTCAGCC-3 (SEQ ID NO: 9)

HincKll HincKll

反向引子 (IF -R1) : Reverse primer (IF -R1):

5， -CCGCT0GAGCAGGTTTCTGGAGGAAGA-3 (SEQ ID NO: 10)。 5, -CCGCT0GAGCAGGTTTCTGGAGGAAGA-3 (SEQ ID NO: 10).

Xhol 将 PCR反应产物以洋菜胶电泳 (agarose electrophoresis)分析确认产物片段大小，接着以 DNA纯化套组 (台湾波仕特公司)进行 PCR产物纯化。再分别将纯化后的 PCR产物以及表现载体 _PET20b以限制酶 Hin Kll以及 Xhol进行酶切后，再以 DNA纯化套组 (台湾波仕特公司)纯化酶切后的 PCR产物及表现载体，接着进行接合反应，将 PCR产物选殖到 _PET20b载体中形成 pET20b-IFNal质体，并将该质体转殖 (transformation)至表现宿主大肠杆菌(£ co ）中，选出带有 pET20b-IF al质体的大肠杆菌，并进行定序确认增殖的 PCR产物序列确实为猪干扰素 al基因 (P IFNal) , 猪干扰素 al基因序列如 SEQ ID No : 1所示，其氨基酸序列如 SEQ ID No: 2所示。 Xhol The PCR reaction product was analyzed by agarose electrophoresis to confirm the size of the product fragment, and then the PCR product was purified by a DNA purification kit (Tai Boss Corporation). The purified PCR product and the expression vector _P ET20b were digested with restriction enzymes Hin K11 and Xhol, respectively, and then the PCR product and expression vector were purified by DNA purification kit (Tai Boss Corporation). The ligation reaction was carried out, and the PCR product was cloned into the _P ET20b vector to form the pET20b-IFNal plastid, and the plastid was transformed into the expression host E. coli (£ co ), and the pET20b-IF al was selected. The plastid Escherichia coli, and the sequence of the PCR product confirmed by sequencing is indeed the porcine interferon al gene (P IFNal), the porcine interferon al gene sequence is shown as SEQ ID No: 1, and the amino acid sequence thereof is SEQ ID No. : 2 is shown.

«例二猪免疫球蛋白 IgG- Fc 4a片因选殖 «Example 2 Porcine immunoglobulin IgG- Fc 4a tablets due to colonization

取,讓猪脾脏，并以 GTC法萃靈。接着以 RT-PCR进行 cDNA合成；取 20 μΐ 总靈、 8 μΐ 1. 25mM d TP以及 1 μΐ 3' 端互补引子，于 70。C作用 5 中后置于冰上，力口入 1 μΐ 灭菌水、 8 μΐ 5xAMV RT buffer, 1 μΐ RNasin和 1 μΐ AMV反转录酶，并置于 42。C作用 1小时，以进行 cDNA合成； |冬合的 cDNA进行 PCR ，在 S ^管中力口入 1 μΐ cDNA、 5 μΐ 10x PCR反应液、 8 μΐ 1. 25 mM dNTP, 1 μΐ 5' 端正向引子、 1 μΐ 3' 端反向引子、 33 μΐ灭菌水、 1 μΐ Taq聚合酶，混合均匀后放入 PCR反应器中 (Applied Biosystems GeneAmp PCR system 2400)，反应条件为先以 95。C 5 中将 DNA变性，接着以 95°C 1 中、 55°C 30秒、 72°C 30秒行30个循环，最后以 72°C 5 中完 j¾PCR反应。其中，猪免疫球蛋白 IgG-Fc 4a片段基因的特异性引子序列如下： Take the pig spleen and extract it with the GTC method. Next, cDNA synthesis was carried out by RT-PCR; 20 μΐ total, 8 μΐ 1. 25 mM d TP and 1 μΐ 3' complementary primer were used at 70. After C, 5 was placed on ice, and 1 μΐ sterile water, 8 μΐ 5×AMV RT buffer, 1 μΐ RNasin and 1 μΐ AMV reverse transcriptase were placed in the mouth. C for 1 hour for cDNA synthesis; | conjugated cDNA for PCR, 1 μΐ cDNA in 5 μM, 5 μΐ 10x PCR reaction solution, 8 μΐ 1. 25 mM dNTP, 1 μΐ 5' The primer, 1 μΐ 3' reverse primer, 33 μΐ sterile water, 1 μΐ Taq polymerase were mixed and placed in a PCR reactor (Applied Biosystems GeneAmp PCR system 2400) under the reaction conditions of 95. The DNA was denatured in C 5 , followed by 30 cycles of 95 ° C 1 , 55 ° C for 30 seconds, and 72 ° C for 30 seconds, and finally the reaction was completed at 72 ° C 5 . Among them, the specific primer sequence of the porcine immunoglobulin IgG-Fc 4a fragment gene is as follows:

正向引子 (IgG-Fl) _: Forward primer (IgG-Fl) _:

5'

(SEQ ID NO: 12) BaMl 5'

(SEQ ID NO: 12) BaMl

反向引子 (IgG-R) _: Reverse primer (IgG-R) _:

5， -CCCAAGCTTTTTACCCGGAGTC-3 ' (SEQ ID NO: 13)。 5, -CCCAAGCTTTTTACCCGGAGTC-3 ' (SEQ ID NO: 13).

Hindlll Hindlll

将 PCR反应产物以洋菜胶电泳分析确认产物片段大小，接着以 DNA纯化套组 (台湾 Geneaid 公司)进行 PCR产物纯化。再分别将纯化后的 PCR产物以及表现载体 pET20b以限制酶 1以及 Hindlll进行酶切反应后，再以 DNA纯化套组 (台湾 Geneaid公司)纯化酶切后的 PCR产物及表现载体，接着进行接合反应，将 PCR产物选殖到 pET20b载体中形成 pET20b-IgG-Fc 4a质体，并将该质专殖至表现宿主大肠杆菌 c^ )中，选出带有 pET20b-IgG-Fc 4a质体的大肠杆菌，并进行定序确认增殖的 PCR产物序列确实为猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a)，猪免疫球蛋白 IgG-Fc 4a片段基因列如 SEQ ID No : 3所示，其氨基酸序列如 SEQ ID No : 4所示。雄例三猪融合 S靈干観 P IFN-Fc) DNA序列的构筑 The PCR reaction product was analyzed by gel electrophoresis to confirm the size of the product fragment, and then the PCR product was purified by a DNA purification kit (Geneaid, Taiwan). The purified PCR product and the expression vector pET20b were digested with restriction enzyme 1 and Hindlll, respectively, and then the PCR product and expression vector were purified by DNA purification kit (Geneaid, Taiwan), followed by ligation reaction. The PCR product was cloned into the pET20b vector to form the pET20b-IgG-Fc 4a plastid, and the substance was colonized into the expression host E. coli c^), and the large intestine with pET20b-IgG-Fc 4a plastid was selected. The PCR product sequence of the bacillus, which is confirmed to be proliferating, is indeed the porcine immunoglobulin IgG-Fc 4a fragment gene (P IgG-Fc 4a), and the porcine immunoglobulin IgG-Fc 4a fragment gene is as shown in SEQ ID No: 3 The amino acid sequence thereof is shown as SEQ ID No: 4. Male case three pig fusion S Linggan 観 P IFN-Fc) DNA sequence construction

在本实施例中，将实施例一所得的猪干扰素 αΐ基因 (P IFNal) (SEQ ID No : 1)以及实施例二所得的猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a) (SEQ ID No: 3)以甘氨酸 (Glycine, G) 及丝氨酸 (Serine, S) j¾的连接子 (1 inker)的 DNA序列 (SEQ ID No： 5)连接，以构筑猪融合重组型干扰素 (P IFNal-Fc 4a)的 DNA序列 (SEQ ID No: 7)。 In the present example, the porcine interferon alpha gene (P IFNal) (SEQ ID No: 1) obtained in Example 1 and the porcine immunoglobulin IgG-Fc 4a fragment gene obtained in Example 2 (P IgG-Fc 4a) were used. (SEQ ID No: 3) is ligated with the DNA sequence (SEQ ID No: 5) of a linker (1 inker) of glycine (Glycine, G) and serine (Serine, S) j3⁄4 to construct a porcine fusion recombinant interferon DNA sequence of (P IFNal-Fc 4a) (SEQ ID No: 7).

首先，以 PCR反应分别增幅猪干扰素 al基因 (P IFNal) (SEQ ID No : 1)以及猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a) (SEQ ID No : 3)，并利用 PCR引子设计将甘氨酉氨酸 j¾的连接子的 DNA序列 (SEQ ID No : 5)分段与猪干扰素 al基因 (P IFNal)以及猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a)—同进行 PCR 增幅。其中，猪干扰素 al基因 (P IFNal)的特异性引子序列如下：正向引子（IF -F2) : First, the porcine interferon al gene (P IFNal) (SEQ ID No: 1) and the porcine immunoglobulin IgG-Fc 4a fragment gene (P IgG-Fc 4a) (SEQ ID No: 3) were respectively increased by PCR reaction, and The DNA sequence of the linker of glycine proline j3⁄4 (SEQ ID No: 5) was segmented with the porcine interferon al gene (P IFNal) and the porcine immunoglobulin IgG-Fc 4a fragment gene (P IgG) using PCR primer design. -Fc 4a) - same as PCR amplification. Among them, the specific primer sequence of the porcine interferon al gene (P IFNal) is as follows: Forward primer (IF-F2):

5' -GCfij ¾7CVTGGCCCCAACCTC-3， (SEQ ID NO: 13) 5' -GCfij 3⁄47CVTGGCCCCAACCTC-3, (SEQ ID NO: 13)

coRV coRV

反向引子 (IF -R2) : Reverse primer (IF -R2):

5' - 愿 CCTGAGCCACCCAGGTTTCTGGAGG- 3 ' (SEQ ID NO: 14)； 5' - Wish CCTGAGCCACCCAGGTTTCTGGAGG-3 ' (SEQ ID NO: 14);

BaiMl 连接子部份序列 BaiMl linker partial sequence

而猪免疫球蛋白 IgG-Fc片段基因的特异性引子序列如下： The specific primer sequences of the porcine immunoglobulin IgG-Fc fragment gene are as follows:

正向引子 (IgG_F2) _: 5， -CG^7ra^TGGAGGCGGAAGCGGCGGTGGAGGATCAGGAACAAAGA-3^> Forward primer (IgG_F2) _: 5, -CG^7ra^TGGAGGCGGAAGCGGCGGTGGAGGATCAGGAACAAAGA-3 ^>

BaiMl 连接子部份序列 (SEQ ID NO: 15) 反向引子 (IgG-R) _: BaiMl linker partial sequence (SEQ ID NO: 15) Reverse primer (IgG-R) _:

5' -CCCAAGCTTTTTACCCGGAGTC-3 ' (SEQ ID NO: 12)。 5'-CCCAAGCTTTTTACCCGGAGTC-3 ' (SEQ ID NO: 12).

Hindlll Hindlll

¾PCR 管中力口入 1 μΐ 100倍稀释的质体 DNA (pET20b_IF a质体或 pET20b-IgG_Fc质体)、 5 μΐ 10x PCR 液、 8 μΐ 1. 25 mM d TP、 1 μΐ 5' 端正向引子、 1 μΐ 3' 端反向引子、 33 μΐ灭菌水、 1 μΐ Taq聚合酶，混合均匀后方 PCHSJ^Z器中（Applied Biosystems GeneAmp PCR system 2400)，反应条件为先以 95。C 5 中将 DNA变性，接着以 95。C 1 中、 55。C 30秒、 72。C 30秒进行 30个循环，最后以 72。C 5 中完 jtPCR反应。 3⁄4 PCR tube with 1 μΐ 100-fold diluted plastid DNA (pET20b_IF a plastid or pET20b-IgG_Fc plastid), 5 μΐ 10x PCR solution, 8 μΐ 1. 25 mM d TP, 1 μΐ 5' positive primer 1 μΐ 3′ end reverse primer, 33 μΐ sterile water, 1 μΐ Taq polymerase, mixed in a uniform PCHSJ^Z apparatus (Applied Biosystems GeneAmp PCR system 2400), and the reaction conditions were 95. DNA is denatured in C5, followed by 95. C 1 medium, 55. C 30 seconds, 72. C 30 seconds for 30 cycles, and finally 72. The jtPCR reaction was completed in C 5 .

将 PCR反应产物以洋菜胶电泳分析确认产物片段大小，接着以 DNA纯化套组 (台湾 Geneaid 公司)进行 PCR产物纯化。翔冬纯化后的 PCR产物猪干扰素 α基因 (P IFNal)以限制酶 BccR 以及 BaOil进行酶切反应，将纯化后的 PCR产物猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a)以限 BaiMl Hindlll进行酶切反应，并将表现载体 pET20b以限制酶^ RV以及〃 il进行酶切反应后，再以 DNA纯化套组 (台湾 Geneaid公司)纯化酶切后的 PCR产物及表现载体，接着进行接合反应，将 PCR产物选殖到 _PET20b载体中形成 pET20b-IF al-Fc 4a质体，并将该质体转殖至表现宿主大肠杆菌 (£ ）中，选出带有 pET20b-IFNal-Fc 4a质体的大肠杆 The PCR reaction product was analyzed by gel electrophoresis to confirm the size of the product fragment, and then the PCR product was purified by a DNA purification kit (Geneaid, Taiwan). The purified PCR product porcine interferon alpha gene (P IFNal) was digested with restriction enzymes BccR and BaOil, and the purified PCR product porcine immunoglobulin IgG-Fc 4a fragment gene (P IgG-Fc 4a) was purified. The restriction enzyme digestion reaction was carried out with BaiMl Hindlll, and the expression vector pET20b was digested with restriction enzymes RV and il il, and then the PCR product and expression vector were purified by DNA purification kit (Geneaid, Taiwan). Following the ligation reaction, the PCR product was cloned into the _P ET20b vector to form the pET20b-IF al-Fc 4a plastid, and the plastid was transformed. Selecting a large intestine with pET20b-IFNal-Fc 4a plastid into the host E. coli (£)

确认增殖的 PCR产物序列确实为本实施例猪融合重组型干扰素 (P IF al-Fc 4a)的 DNA序列 (SEQ IDThe PCR product sequence confirming proliferation was indeed the DNA sequence of the porcine fusion recombinant interferon (P IF al-Fc 4a) of the present example (SEQ ID

No: 7)，而本实施例猪融合重组型干扰素 (P IFNal-Fc 4a)的氨基酸序列如 SEQ ID No: 8麻。 «例四其它猪融合重干 ift^(P IFNa-Fc) DNA序列的构筑 No: 7), and the amino acid sequence of the porcine fusion recombinant interferon (P IFNal-Fc 4a) of the present example is SEQ ID No: 8 hemp. «Example 4 Other porcine fusion heavy dry ift^(P IFNa-Fc) DNA sequence construction

除了实施例一所得的猪干扰素 al基因 (P IF al) (SEQ ID No: 1)之外，猪干扰素 a还有 a2至 al7等亚型（subtype) , 猪干扰素 a2至 al7的 DNA序列分别如 SEQ ID Nos : 16、 18、 20、 22、 24、 26、 28、 30、 32、 34、 36、 38、 40、 42、 44及 46所示，其¼¾酉辨列则分别如 SEQ ID Nos： 17、 In addition to the porcine interferon al gene (P IF al) (SEQ ID No: 1) obtained in Example 1, porcine interferon a also has subtypes of a2 to al7, and porcine interferon a2 to al7 DNA. The sequences are as shown in SEQ ID Nos: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46, respectively. ID Nos: 17,

19、 21、 23、 25、 27、 29、 31、 33、 35、 37、 39、 41、 43、 45及 47麻。 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47 hemp.

此外，除了实施例二所得的猪免疫球蛋白 IgG-Fc 4a片段基因 (P IgG-Fc 4a) (SEQ ID No: 3) 之外，本实施例并以猪免疫球蛋白 IgG-Fc la、 lb、 2a、 2b及 4a片段基因 (P IgG-Fc la, lb, 2a, 2b, 4a)分别与 ¾猪干扰素 al至 al7的 DNA序列构筑形成猪融合重组型干扰素。猪免疫球蛋白 IgG-Fc la, lb、 2a、 2b及 4a片段基因 (P IgG-Fc la, lb, 2a, 2b, 4a)的 DNA序列分别如 SEQ ID Nos : 48、 50、 52、 54及 3所示，其¼¾酉辨列则分别如 SEQ ID Nos： 49、 51、 53、 55及 4所示。 Further, in addition to the porcine immunoglobulin IgG-Fc 4a fragment gene (P IgG-Fc 4a) obtained in Example 2 (SEQ ID No: 3), the present example is porcine immunoglobulin IgG-Fc la, lb The 2a, 2b and 4a fragment genes (P IgG-Fc la, lb, 2a, 2b, 4a) were constructed with the DNA sequence of 3⁄4 porcine interferon al to al7, respectively, to form a porcine fusion recombinant interferon. The DNA sequences of the porcine immunoglobulin IgG-Fc la, lb, 2a, 2b and 4a fragment genes (P IgG-Fc la, lb, 2a, 2b, 4a) are SEQ ID Nos: 48, 50, 52, 54 and As shown in Fig. 3, the discrimination is shown in SEQ ID Nos: 49, 51, 53, 55 and 4, respectively.

同实施例三腿，分别将猪干扰素 αΐ至 α17基因 (P IFNal-al7) (SEQ ID Nos : 1、 16、 18、 In the same three legs of the example, porcine interferon αΐ to α17 gene (P IFNal-al7) (SEQ ID Nos: 1, 16, 18,

20、 22、 24、 26、 28、 30、 32、 34、 36、 38、 40、 42、 44及 46)以及猪免疫球蛋白 IgG-Fc la, lb、 2a、 2b及 4a片段基因 (P IgG-Fc la, lb, 2a, 2b, 4a) (SEQ ID Nos : 48、 50、 52、 54及 3) 以德酸 (G)及丝氨酸 (S)组成的连接子的 DNA序列 (SEQ ID No: 5)连接，以构筑形成各种猪融合重组型干扰素 (P IF a-Fc)的 DNA序列 (SEQ ID Nos : 7、 56、 58、 60、 62、 64、 66、 68、 70、 72、 74、 76、 78、 80、 82、 84、 86、 88、 90、 92、 94、 96、 98、 100、 102、 104、 106、 108、 110、 112、 114、 116、 118、 120、 122、 124、 126、 128、 130、 132、 134、 136、 138、 140、 142、 144、 146、 148、 150、 152、 154、 156、 158、 160、 162、 164、 166、 168、 170、 172、 174、 176、 178、 180、 182、 184、 186、 188、 190、 192、 194、 196、 198、 200、 202、 204、 206、 208、 210、 212、 214、 216、 218、 220及 222)，并以 DNA合成仪 (应用生物***公司，美国)合成上述各种猪融合重组型干扰素 (P IFNa-Fc)的 DNA序列；合成 DNA时，并于各猪融合重组型干扰素 (P IFNa-Fc)的 DNA序列 5' 端前加上一个 ^ cRV (GATATC)限制酶切位，以及在各猪融合重组型干扰素 (P IF a-Fc)的丽序列 3，端后加上一个〃 I I (MGCTT)限制酶切位。 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46) and porcine immunoglobulin IgG-Fc la, lb, 2a, 2b and 4a fragment genes (P IgG -Fc la, lb, 2a, 2b, 4a) (SEQ ID Nos: 48, 50, 52, 54 and 3) DNA sequence of a linker consisting of deacid (G) and serine (S) (SEQ ID No: 5) ligation to construct a DNA sequence for the formation of various porcine fusion recombinant interferons (P IF a-Fc) (SEQ ID Nos: 7, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, and 222) DNA synthesizer System Company, USA) Synthesizes the DNA sequences of the various porcine fusion recombinant interferons (P IFNa-Fc); when synthesizing DNA, and merging the 5' end of the DNA sequence of recombinant interferon (P IFNa-Fc) in each pig Add a ^ cRV (GATATC) restriction enzyme cleavage site, and add a 〃 II (MGCTT) restriction enzyme cleavage site in the porcine sequence 3 of each pig fusion recombinant interferon (P IF a-Fc).

将 5' 端及 3' 端分别带有 ¾cRV以及〃 οΙΙΙ限制酶切位的合的各种猪融合重组型干扰素 (P IFNa-Fc)的 DNA以及表现载体 pSecTag2 (B)分别以限制酶 ccRV以及〃 οΐ II进行酶切反应，再以 DNA纯化套组 (台湾 Geneaid公司)纯化酶切后的 DNA片段及表现载体，接着进行接合反应，将各 DNA片誕殖到 _PSecTag2 (B)载体中形成各种 _PSecTag2 (B) -IFNa-Fc质体，并将该质#$专殖至表现宿主大肠杆菌 (£ _C0 )中，选出带有各种 pSe_CTag2 (B) -IFNa-Fc质体的大肠杆菌，各猪融合重组型干扰素 (P IFNa-Fc)的 S酉辨列分别如 SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、The porcine fusion recombinant interferon (P IFNa-Fc) DNA and the expression vector pSecTag2 (B) with the 3'4RR and the 〃οΙΙΙ restriction enzyme cleavage at the 5' and 3' ends, respectively, with the restriction enzyme ccRV And 〃οΐ II was subjected to enzymatic cleavage reaction, and the DNA fragment and the expression vector were purified by DNA purification kit (Geneaid, Taiwan), followed by ligation reaction, and each DNA piece was incubated into _P SecTag2 (B) vector. Various _P SecTag2 (B) -IFNa-Fc plastids were formed, and the cytoplasmic ## was colonized into the expression host E. coli (£ _C0 ), and various pSe _C Tag2 (B) -IFNa-Fc were selected. For the plastid Escherichia coli, the S酉 of each porcine fusion recombinant interferon (P IFNa-Fc) is identified as SEQ ID Nos: 8, 57, 59, 61, 63, 65, 67, respectively.

69、 ¹ 71、 73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、皿、 103、 105、 107、69, ¹ 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, Dish, 103, 105, 107,

109、 m、 113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、109, m, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,

143、 145、 147、 149、 151、 153、 155、 157、 159、 16U 163、 165、 167、 169、 171、 173、 175、143, 145, 147, 149, 151, 153, 155, 157, 159, 16U 163, 165, 167, 169, 171, 173, 175,

177、 179、 18K 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、177, 179, 18K 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,

211、 213、 215、 217、 219、 221及 223所示。 «例五猪融合干 ift¾(P IFNa- Fc)的表现 211, 213, 215, 217, 219, 221, and 223 are shown. «Example 5 pig fusion dry ift3⁄4 (P IFNa- Fc) performance

先将实施例四所得到的 pSecTag2 (B) -IFNal-Fc 4a质体转染 (transfection)至中国仓鼠卵巢细胞株 (CH0 cells)。取 4 (ig _PSecTag2 (B) -IFNal-Fc 4a质体 DNA加入无抗生素且无血清的 VP 培养基（Invitrogen)中震荡 15秒（混合液 A) ; 另外将 4 g Lipofectamine试剂（Invitrogen)加入生素且 ¾ώ清的 VP培养基中 (混合液 B)，于室温下作用 5 中；接着将混合液 A加入混合液 B中，震荡 15秒后于 37。C下作用 20 中。 ^上述混合液 (A+B)均匀加入 ¾ΰ 细胞中，将细謹于 37。 (：、 5 % C0₂培养箱内作用 6小时后，去除混合液并加入含有 10 %胎牛血清 (FBS)的 F12培养基，将细胞置于 37° (：、 5 % C0₂培养箱内继续培养 48小时。 The pSecTag2(B)-IFNal-Fc 4a plastid obtained in Example 4 was first transfected into Chinese hamster ovary cell line (CH0 cells). 4 (ig _P SecTag2 (B) - IFNal-Fc 4a plastid DNA was added to antibiotic-free and serum-free VP medium (Invitrogen) for 15 seconds (mixture A); 4 g of Lipofectamine reagent (Invitrogen) was added. In the VP medium (mixture B), which is phlegm and 3⁄4 ώ clear, is applied to 5 at room temperature; then the mixture A is added to the mixture. In liquid B, it was shaken after 15 seconds at 37. Under C, it works in 20. ^ The above mixture (A + B) is evenly added to the 3⁄4 ΰ cells, which will be fine at 37. (:, after 6 hours in a 5 % C0 ₂ incubator, remove the mixture and add F12 medium containing 10% fetal bovine serum (FBS) and place the cells in a 37° (:, 5% C0 ₂ incubator) Continue to culture for 48 hours.

接着，以 Zeocin ½素筛选带有猪融合重组型干扰素基因的 CH0细胞。将经过转染的 CH0 细胞株继代培养于 24孔细胞培养盘中，以含有 10% FBS、 100 Units/ml Penicillin, 100 Units/ml Streptomycin和 700 μ_§/ιιι1 Zeocin抗生素的 F12培养基培养以进行筛选。接着将细胞以磷酸盐缓冲液 (PBS)清洗两次后加入 0. 125%胰蛋白酶 (Trypsin)进行消化，待细胞圆化后，摇晃角瓶使细胞脱落，以培养 ¾冬细胞冲营悬浮，细胞培养于 37。 (：、 5% C0₂培养箱，继代两次后，待活着的细胞约剩一至两成时，将细胞培养基替换成含有 50 g/ml Zeocin抗生素以及 10% FBS的 F12培养基，待细胞恢复原来生长速度后，以间接免疫荧光染色 (IFA)法、鍾遊吉免疫分析 (ELISA)法以及西方墨点法确认细胞是否带有猪融合重组型干扰素 (P IF al-Fc 4a)基因并且表现该重组蛋白。 Next, CH0 cells harboring the porcine fusion recombinant interferon gene were screened with Zeocin. The transfected cell line CH0 is subcultured in 24-well cell culture dishes containing 10% FBS, 100 Units / ml Penicillin, 100 Units / ml Streptomycin and 700 μ _§ / ιιι1 Zeocin antibiotic to the culture medium F12 Screening. Then, the cells were washed twice with phosphate buffered saline (PBS) and then digested with 0.15% trypsin (Trypsin). After the cells were rounded, the cells were shaken by shaking the flasks to culture the 3⁄4 winter cells. The cells were cultured at 37. (:, 5% C0 ₂ incubator, after subculture twice, when there are about one to two percent of cells to be alive, replace the cell culture medium with F12 medium containing 50 g/ml Zeocin antibiotic and 10% FBS. After the cells were restored to their original growth rate, they were confirmed by indirect immunofluorescence staining (IFA), Zhongyouji immunoassay (ELISA), and western blotting to confirm whether the cells contained the porcine fusion recombinant interferon (P IF al-Fc 4a) gene. And the recombinant protein is expressed.

1. 以间接免疫荧光譲 IFA)法翻赌融合重醒干擬的麵 1. Indirect immunofluorescence (IFA) method to gamble and reinvigorate the face

将转染的 CH0细胞接种在 24孔细胞培纖内 (1 X 10⁵ cells/well)至细胞长到约 8-9 茜时，以 PBS清洗两次后加入 80%丙酮 (-20°C)，并于 4。C下静置 30 中进 fi¾田胞固定，再以 PBS清洗三次后，加入以 PBS稀释 1, 000倍的 rabbit anti Porcine IgG- FITC antibody (300 μΐ/well) 置于 37°C培养箱中¾¾作用 30 中，再以 PBS清洗三次后，每孔加入 250 μΐ PBS，最后以荧光显微镜观察。 The transfected CH0 cells were seeded in 24-well cell culture medium (1×10 ⁵ cells/well) until the cells grew to about 8-9 ,, washed twice with PBS and then added with 80% acetone (-20 ° C). , and at 4. After standing at 30, the cells were fixed in fi3⁄4 cells, washed three times with PBS, and then added to a rabbit anti-porcine IgG- FITC antibody (300 μΐ/well) diluted 1 000 times in PBS and placed in a 37 ° C incubator 3⁄43⁄4 In action 30, after washing three times with PBS, 250 μM PBS was added to each well, and finally observed with a fluorescence microscope.

荧光显微镜观察结果如图 1麻。经过 Zeocin抗生素筛选后存活的 CH0细胞带有猪融合重组型干扰素基因，以间接免疫荧光染色 (IFA)法分析可观察到荧光讯号 (如图 1A麻)；而只有转染 _PSecTag2 (B)载体的 CH0细胞则没有荧光讯号 (如图 1B麻)。 The results of fluorescence microscopy are shown in Figure 1. CH0 cells surviving after Zeocin antibiotic screening were porcine fusion recombinant interferon gene, and fluorescent signals were observed by indirect immunofluorescence staining (IFA) analysis (Fig. 1A); only transfected _P SecTag2 (B) The carrier's CH0 cells have no fluorescent signal (as shown in Figure 1B).

2. 以« ^免疫分析 (ELISA)法翻赌融合重醒干擬的麵 2. Use the « ^ immunoassay ( ELISA ) method to gamble and reinvigorate the face

将 I X 10⁵颗待检测细胞接种于 25平方公分细胞培养瓶 (25T_flask)中，以含有 10% FBS的 F-12 培养基培养 72小时后，取其细胞上层培养基以 ELISA coating buffer (0. 1 M NaHC0₃以及 0. 1 M N¾C0₃ pH9. 6)进行 2、 4、 8、 16、 32、 64、 128倍稀释， ^^样 :φ¾Π00 μΐ涂布 (coating)于 ELISA plate (NUNC)中，置于 4。C下作用 24小时后，去除上清液，以 ELISA washing buffer (0. 9% NaCl, 0. 1 % Tween20)清洗三次，力口入 100 μΐ blocking buffer (1% BSA in ELISA washing buffer) , 于室温作用 1小时以去除非特异性反应，随后去除 blocking buffer并以 ELISA washing buffer 清 ί¾≡次，力口入 100 μΐ mouse anti IF a单株抗体 (SANTA CRUZ)，该抗体事先以含有 1% BSA的 ELISA washing buffer稀释 500倍；于室温作用 1小时后，以 ELISA washing buffer清洗 6次，力口入 100 μΐ已 ¾Η己辣 ¾31氧化物酶 (HRP)的 goat anti mouse抗体 (KPL)，该抗体事先以含有 1% BSA 的 ELISA washing buffer稀释 1, 000倍；于室温作用 1小时后，以 ELISA washing buffer清洗六次，加入 100 μΐ 受质 3, 3' , 5, 5' -四甲基联苯胺二盐酸（3, 3' , 5, 5' -tetramethylbenzidine, TMB) (KPL)呈色 10 中后，再以 ELISA reader在吸光值波长 (0. D) 650皿下读取吸光值。 The IX 10 ⁵ cells to be tested were seeded in a 25 cm 2 cell culture flask (25T_flask), cultured in F-12 medium containing 10% FBS for 72 hours, and then the cell upper medium was taken as an ELISA coating buffer (0. 1 M NaHC0 ₃ and 0.1 M N3⁄4C0 ₃ pH9. 6) 2, 4, 8, 16, 32, 64, 128-fold dilution, ^^-like: φ3⁄4Π00 μΐ coating in ELISA plate (NUNC) , placed at 4. After 24 hours of action at C, the supernatant was removed, washed three times with ELISA washing buffer (0.9% NaCl, 0.1% Tween20), and 100 μΐ blocking buffer (1% BSA in ELISA washing buffer) was applied. The reaction was allowed to stand at room temperature for 1 hour to remove the non-specific reaction, then the blocking buffer was removed and ELISA washing buffer was used to remove 100 μΐ mouse anti IF a monoclonal antibody (SANTA CRUZ), which was previously contained in 1% BSA. The ELISA washing buffer was diluted 500 times; after 1 hour at room temperature, it was washed 6 times with ELISA washing buffer, and 100 μΐ of goat anti-antibody (HRP) goat anti mouse antibody (KPL) was prepared. Dilute 1 000 times with ELISA washing buffer containing 1% BSA; after 1 hour at room temperature, wash 6 times with ELISA washing buffer, add 100 μΐ of the receptor 3, 3', 5, 5'-tetramethylbenzidine After the (3, 3', 5, 5'-tetramethylbenzidine, TMB) (KPL) color was 10, the absorbance was read by an ELISA reader at the absorbance wavelength (0. D) 650.

以 ELISA方法检测细胞分泌物含量的结果如图 2所示，带有猪融合重组型干扰素基因的 CH0 细胞分泌物稀释至 128倍后仍可测到高量的猪融合重组型干扰素，但带有 _PSecTag2 (B)载体的 CH0 细胞，其细胞培养基中则测不到猪融合重组型干扰素。 The results of ELISA assay for cell secretions are shown in Figure 2. High levels of porcine fusion recombinant interferon can still be detected after dilution of CH0 cell secretion with porcine fusion recombinant interferon gene to 128-fold. In CH0 cells bearing the _P SecTag2 (B) vector, porcine fusion recombinant interferon was not detected in the cell culture medium.

3. 以西方墨点 ¾^测猪融合 sis pi^的表现 3. Measure the performance of pig fusion with Western ink points 3⁄4^

取上述细胞上层培养基以十二垸基磺酸钠-聚丙烯酰胺胶电泳分析 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)后，将胶体上的蛋白转印至 PVDF膜上，并将转印后的 PVDF J3IS于 blocking buffer (5% Skim milk, in TBST)中，于 4。C作用 16-24 小时，以去除非特异性反应，再以 TBST (10 mM Tris-HCl pH 8. 0, 150 mM NaCl, ( 清 ¾¾≡次，每次五她随后加入瞧 e anti IFNa单株抗体 (SANTA CRUZ)，该抗体事先以含有 0. 5% skin milk的 TBST稀释 500倍，于室温下轻轻摇晃作用 1小时后，以 TBST清洗六次，每次 5 中，再加入已 ¾Η己碱性磷酸酶 (AP)的 goat anti mouse抗体，该抗体事先以含有 0. 5% skin milk 的 TBST稀释 2000倍，于室温下轻轻摇晃作用 1小时后，以 TBST清洗 6次，每次 5 中，再加 AP受质 NBT/BCIP (Bio-Rad)呈色约 5 中后，倒掉显色剂以清水冲洗终 ± 色反应。另外也使用已己碱性磷酸酶 (AP)的 goat anti-Porcine IgG的抗体 (KPL)以及已 fe己碱性磷酸酶 (AP)的 mouse anti 6 X His单株抗体（invitrogen)侦测猪融合重组型干扰素。 After the above-mentioned cell upper culture medium is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein on the colloid is transferred onto the PVDF membrane, and The transferred PVDF J3IS is in blocking buffer (5% Skim milk, in TBST), at 4. C role 16-24 Hours to remove non-specific reactions, followed by TBST (10 mM Tris-HCl pH 8.0, 150 mM NaCl, (3⁄43⁄4 times, each time five she subsequently added 瞧e anti IFNa monoclonal antibody (SANTA CRUZ), The antibody was diluted 500-fold with TBST containing 0.5% skin milk in advance, and gently shaken at room temperature for 1 hour, then washed six times with TBST, 5 times each time, and then added 3⁄4 Η alkaline phosphatase (AP). Goat anti mouse antibody, which was diluted 2000 times with TBST containing 0.5% skin milk, gently shaken at room temperature for 1 hour, then washed 6 times with TBST, 5 times each time, plus AP After the NBT/BCIP (Bio-Rad) color is about 5, the developer is rinsed off with water to wash the final color reaction. The antibody of goat anti-Porcine IgG with alkaline phosphatase (AP) is also used. KPL) and mouse anti 6 X His monoclonal antibody (invitrogen), which has been tested with porcine alkaline phosphatase (AP), detects porcine fusion recombinant interferon.

西方墨点¾ ^析结果如图 3所示，带有猪融合重组型干扰素基因的 CH0细胞分泌物可被 mouse anti IFNa 抗体、 goat anti-Porcine IgG的抗体 (KPL)以及 mouse anti 6 X His单株抗体辨认，显示该细胞分泌物含有猪融合重组型干扰素。 Western blotting results are shown in Figure 3. The CH0 cell secretion with the porcine fusion recombinant interferon gene can be used by mouse anti IFNa antibody, goat anti-Porcine IgG antibody (KPL), and mouse anti 6 X His. Individual antibody recognition revealed that the cell secretion contained porcine fusion recombinant interferon.

«例六猪融合干 ift¾(P IFN_a— Fc)的小及 ^»^，以及以 PRRS检测该融合重醒干撤灘生 «Example six pig fusion dryift3⁄4 (P IFN _a - Fc) small and ^^^, and PRRS detection of the fusion rejuvenation dry beach

1. 猪融合重醒干観 P IFNa- Fc)的小 M ^制程 1. Pig fusion rejuvenation dry P IFNa- Fc) small M ^ process

将带有猪融合重组型干扰素 (P INFal-Fc 4a)基因 (SEQ ID No: 7)的 CH0细胞以 2 X 10⁶颗细胞数接种培养于 25平方公分细胞培养瓶 (25T-flask)中，并以含有 10% FBS以及 100 Units/ml Penicillin和 100 Units/ml Streptomycin的 F12培养基培养 24小时后，去除原培养基，以 PBS 清洗一次后，加入含有 100 Units/ml Penicillin和 100 Units/ml Streptomycin的 CHO-S-SFM II ¾1清培养基 (GIBC0)，于更换为¾1清培养基后，分别于每隔 24小时、 48小时、 72小时三种不同的时间间隔更换新的无血清培养基（含有 100 Units/ml Penicillin 和 100 Units/ml Streptomycin) , 并且收集被更换的细胞生长液，以 1, 000 rpm离心 10 中，将细胞以及细胞碎片去除，取其上清液，即可获得猪融合重组型干扰素。接着，藉由猪生殖和呼吸道综合症病毒 (Porcine reproductive and respiratory syndrome virus, PRRS)以测定该猪融合重组型干扰素的抗病毒活性。 CH0 cells harboring the porcine fusion recombinant interferon (P INFal-Fc 4a) gene (SEQ ID No: 7) were seeded in a 25 square centimeter cell culture flask (25T-flask) at a dose of 2×10 ⁶ cells. After culturing for 24 hours in F12 medium containing 10% FBS and 100 Units/ml Penicillin and 100 Units/ml Streptomycin, the original medium was removed, washed once with PBS, and then added with 100 Units/ml Penicillin and 100 Units/ Mp Streptomycin CHO-S-SFM II 3⁄41 Clear Medium (GIBC0), after replacement with 3⁄41 clear medium, replace the new serum-free culture at three different time intervals every 24 hours, 48 hours, 72 hours Base (containing 100 Units/ml Penicillin and 100 Units/ml Streptomycin), and collect the replaced cell growth solution, centrifuge 10 at 10,000 rpm, remove the cells and cell debris, and take the supernatant to obtain Pig fusion recombinant interferon. Next, the antiviral activity of the porcine fusion recombinant interferon was determined by Porcine reproductive and respiratory syndrome virus (PRRS).

2. 测定猪融合靈干撤離 PRRS病毒的灘 2. Determination of pig fusion Linggan evacuation PRRS virus beach

将上述收获的猪融合重组型干扰素以含有 1% FBS 以及 100 Units/ml Penicillin禾口 100 Units/ml Streptomycin的 MEM培养基进行 10倍、 20倍、 40倍、 80倍、 160倍、 320倍、 640倍、 1, 280倍、 2, 560倍序列稀释。并取 MARC-145细胞接种至 96 ¾έ田胞培 (1. 5Χ 10⁴ ?L)，培养于 37。 (：、 5% C0₂培养箱中，培养 16-24小时待细胞贴附后，移齡咅养基，力口入上述稀释后的猪融合重组型干扰素，样品 ΙΟΟ μ Ι/well, 稀释倍数的样品分别接种于 4个孔中，于 37。 (：、 5% (：0₂恒温培养箱中培养细胞 24小时后，移除上清液，并于每孔加入 100 μ ΐ含有 100 TCIDs^ PRRS病毒；另外，亦有其它不同处理的组别：未攻毒未添加干扰素、有攻毒未添加干扰素、添加干扰素未攻毒处理等其它三组；于 37。 (：、 5% C0₂培养箱中，当只加病毒组的细胞产生 90%以上的细胞病变效应 (cytopathic effects, CPE)时，大约是感染病毒后 120小时，进行干扰素抗病毒活性的测定。首先将细胞上清液移除，以 PBS清洗两次后，力口入 100 μ 1 80%丙酮 (_20。C)，并于 4。C 下固定 30 接着移除丙酮以 PBS ί¾≡次后，力口入 1%氯化甲基玫瑰苯胺 (methylrosaniline chloride)染色 20 中，再以蒸馏水洗 5次，去除未染上细胞的氯化甲基玫瑰苯胺，最后将蒸馏水吸干并加入 100%乙酮冬氯化甲基玫瑰苯胺的颜色溶出来，作用 10 中后，再以 ELISA reader 在吸光值波长 (0. D) 550 nm下读取吸光值，并以下列公式计算干扰素抗病毒活性。 The harvested pig-fused recombinant interferon was 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, 320-fold in MEM medium containing 1% FBS and 100 Units/ml Penicillin and 100 Units/ml Streptomycin. , 640 times, 1, 280 times, 2, 560 times serial dilution. The MARC-145 cells were inoculated to 96 3⁄4 έ field cell culture (1.5 Χ 10 ⁴ LL) and cultured at 37. (:, 5% C0 ₂ incubator, cultured for 16-24 hours after cell attachment, migrating gilt, sputum into the diluted porcine fusion recombinant interferon, sample ΙΟΟ μ Ι / well, diluted Multiples of the samples were inoculated into 4 wells at 37. (:, 5% (: 0 ₂ cultured cells were incubated for 24 hours, the supernatant was removed, and 100 μ ΐ was added to each well containing 100 TCIDs ^ PRRS virus; In addition, there are other different treatment groups: no challenge, no interferon added, no interferon added, no interferon added, and other three groups; 37. (:, 5 In the % C0 ₂ incubator, when only the cells of the virus group produced more than 90% of cytopathic effects (CPE), the antiviral activity of interferon was measured approximately 120 hours after infection. The supernatant was removed. After washing twice with PBS, force 100 μl of 80% acetone (_20.C) and fix it at 4 ° C. Then remove the acetone to PBS ί 3⁄4 times. 1% methylrosaniline chloride stained in 20, followed by distilled water Wash 5 times, remove the chlorinated methyl rose aniline that has not been stained with cells, and finally dilute the distilled water and add 100% ethyl ketone methyl chloromethyl rose aniline to dissolve it. After 10 times, use ELISA reader. Absorbance value (0. D) The absorbance was read at 550 nm and the interferon antiviral activity was calculated by the following formula.

,、，、 ODmaximum + ODminimum ,, ,, ODmaximum + ODminimum

公式 1: = 0D50% IF titer (U ml—¹) = Tn Formula 1: = 0D50% IF titer (U ml— ¹ ) = Tn

公式 2: Formula 2:

[ (Tn+l-Tn) [ (Tn+l-Tn)

OD maximum: 未攻毒未添加干扰素组以及未攻毒添加干扰素组的平均值； OD maximum: the average value of the uninterfered group without interferon plus the interferon group added without attack;

0D minimum: 有攻毒未添加干扰素组的平均值； 0D minimum: the average value of the interferon group without attacking;

Tn: 大于 0D 50%的稀释倍数； Tn: a dilution factor greater than 0% 50%;

Tn+1: 小于 0D 50%的稀释倍数； Tn+1: less than 0D 50% dilution factor;

ODn: 大于 0D 50%的 0D平均值； ODn: 0D average value greater than 0D 50%;

0Dn+l: 小于 0D 50%的 0D平均值。 0Dn+l: 0D average less than 0D 50%.

以小量制程制备的猪融合重组型干扰素，其抗 PRRS病毒活性测定的结果如表一所示。结果显示，小 *»漸制备的猪融合重组型干扰素皆具抗 PRRS病毒活性。 The results of the anti-PRRS virus activity assay of the porcine fusion recombinant interferon prepared by a small amount of the preparation are shown in Table 1. The results showed that the small porcine fusion recombinant interferon was anti-PRRS virus activity.

表一以 25平方公分细胞培养瓶 (25T-flask)生产的猪融合重组型干扰素的抗 PRRS病毒活性 (IU/ml) Table 1 Anti-PRRS virus activity (IU/ml) of porcine fusion recombinant interferon produced in 25 square centimeter cell culture flask (25T-flask)

由产出的猪融合重组型干扰素的单位活性 (结果如表一戶; 乘以 5毫升 (25T-flask收获液总体积)即可得到总活性 (抗 PRRS)，结果如表二所示。表二以 25平方公分细胞培养瓶 (25T- flask)生产的猪融合重组型干扰素的总活隱位： IU/5 ml) The total activity (anti-PRRS) was obtained from the produced pig fusion recombinant interferon unit activity (the results are as shown in Table 1; multiplied by 5 ml (25T-flask total volume). The results are shown in Table 2. Table 2 Total live hidden position of porcine fusion recombinant interferon produced by 25 square centimeter cell culture flask (25T-flask): IU/5 ml)

3. 以转 »咅养^猪融合重干 ift^(P IFNa-Fc) 3. Transfer to 咅 ^ ^ pig fusion and dry ift^ (P IFNa-Fc)

将带有猪融合重组型干扰素 (P INFal-Fc 4a)基因 (SEQ ID No: 7)的 CH0细胞继代培养于 175 平方公分细胞培养瓶 (175T- flask)中，待长满后吸弃原培养基，以 PBS洗细胞二次后，加入 0. 125% 胰蛋白酶 (trypsin)与细胞作用，使细胞由培养瓶表面脱落，并以含有 10% FBS以及 100 Units/ml Penicillin和 100 Units/ml Streptomycin的 F12培养基冲洗培养瓶表面并将脱落的细胞打散，以进行细胞计数。分别于不同的转瓶内接种 6. 8 X 10⁷以及 8 X 10⁷颗细胞，并于每支转瓶内力口入含有 10% FBS的 F12培养基使总 #f只达 200 mL，将细腿于 37。C培养箱中，以转速 6 min/圈培养。待培养 24小时细胞贴附后，去除转瓶内培养基并以 PBS洗细胞 1次，去除 PBS后加入含有 100 Units/ml Penicillin禾卩 100 Units/ml Streptomycin的¾1清培养基 CH0— S— SFM II (Invitrogen) mmm, 于更换为¾！清培养基后，每隔 72小时更换新的 ¾清培养基 (含有 100 Units/ml Penicillin和 100 Units/ml Streptomycin) , 共更换 6次；并且收集被更换的细胞生长液，以 1, 000 rpm离心 10 中，将细胞以及细胞碎片去除，取其上清液测猪融合重组型干扰素抗 PRRS 病毒活性。猪融合重组型干扰素抗 PRRS病毒活性系以 MTT法测定。 CH0 cells harboring the porcine fusion recombinant interferon (P INFal-Fc 4a) gene (SEQ ID No: 7) were subcultured in 175 cm ^ 2 cell culture flasks (175T-flask), and were discarded after they were overgrown. The original medium was washed twice with PBS, and 0.15% of trypsin was added to the cells to remove the cells from the surface of the culture flask, and contained 10% FBS and 100 Units/ml Penicillin and 100 Units/. The F12 medium of ml Streptomycin rinses the surface of the flask and breaks off the shed cells for cell counting. Inoculate 6. 8 X 10 ⁷ and 8 X 10 ⁷ cells in different spinner flasks respectively, and inject F12 medium containing 10% FBS into each flask to make the total #f only 200 mL. Legs at 37. In a C incubator, culture at a speed of 6 min/turn. After 24 hours of cell attachment, the medium in the flask was removed and the cells were washed once with PBS. After removing the PBS, 3⁄41 clear medium CH0-S-SFM containing 100 Units/ml Penicillin and 100 Units/ml Streptomycin was added. II (Invitrogen) mmm, replaced with 3⁄4! After clearing the medium, replace the new 3⁄4 clear medium (containing 100 Units/ml Penicillin and 100 Units/ml Streptomycin) every 6 hours for a total of 6 changes; and collect the replaced cell growth solution at 1,000 rpm. In centrifugation 10, the cells and cell debris were removed, and the supernatant was used to measure the activity of the porcine fusion recombinant interferon against PRRS virus. Porcine fusion recombinant interferon anti-PRRS virus activity was determined by MTT assay.

以转瓶培养生产猪融合重组型干扰素的抗病毒活圆定结果如 ¾≡麻，结果显示，以转瓶生产 (大 ¾ji」穏的猪融合重组型干扰素具抗 PRRS病毒活性，且相较于小 «ji」程，经转瓶生产 (大 m的猪融合重组型干扰素，其抗 PRRS病毒活性更高。以转并胜产的猪融合重组型干扰素的抗 PRRS病毒活性 (IU/ml) The antiviral round results of porcine fusion recombinant interferon produced by spinner flask culture, such as 3⁄4 ramie, showed that the porcine fusion recombinant interferon produced by the spinner flask was resistant to PRRS virus activity, and Compared to the small «ji" process, the production of the bottle (large The pig of m fusion recombinant interferon has higher anti-PRRS virus activity. Anti-PRRS virus activity (IU/ml) of porcine fusion recombinant interferon

«例七猪融合干 ift¾(P IFN_a— Fc)以与猪免; ^蛋白 IgG— Fc片 ¾M合皿的干扰素的抗 1¾1«病# ^生的比较 «Example seven pig fusion dryift3⁄4 (P IFN _a - Fc) to compare with the porcine exemption; ^ protein IgG - Fc tablets 3⁄4M dish of interferon anti-13⁄41 « disease # ^

将丽 C-145细胞以每孔 1. 5 X 10⁴细胞的细胞密度培养 16-24小时，再分别处理：猪融合重组型干扰素 (P IFNal-Fc 4a组)及未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal 组) 16-24小时，再以 PRRS病毒 (100 TCID₅。)接种该细胞，经 4-5天后，以 MTT法判读活性。 The C-145 cells were cultured at a cell density of 1.5×10 ⁴ cells per well for 16-24 hours, and then separately treated: porcine fusion recombinant interferon (P IFNal-Fc 4a group) and non-porcine immunoglobulin The IgG-Fc fragment was fused to the recombinant interferon (P IFNal group) for 16-24 hours, and then the cells were inoculated with PRRS virus (100 TCID ₅ ), and after 4-5 days, the activity was judged by the MTT method.

如 ¾¾¾图 4所示，结果显示，相较于未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal Μ) , 本发明的猪融合重组型干扰素 (P IFNal-Fc 4a迎的抗 PRRS病毒活性较高。表五猪融合重组型干扰素 (P IFNal-Fc 4a组)及未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal 的抗 PRRS活性的比较

As shown in Figure 4, the results show that the porcine fusion recombinant interferon (P IFNal-Fc 4a welcome) of the present invention is compared to the interferon (P IFNal Μ) which has not been fused with the porcine immunoglobulin IgG-Fc fragment. The activity of anti-PRRS virus is higher. Table 5 Comparison of anti-PRRS activity of P IFNal with recombinant interferon (P IFNal-Fc 4a group) and interferon recombinantly fused with porcine immunoglobulin IgG-Fc fragment

«例八猪融合干 ift¾(P IFN_a— Fc)与未与猪免疫球蛋白 IgG— Fc片 ¾Μ合重组的干 (P IFNa)的抗 1¾病# ^生的比较 «Example 8 porcine fusion dryift3⁄4 (P IFN _a - Fc) compared with porcine immunoglobulin IgG- Fc tablets 3⁄4 recombination of dry (P IFNa) anti- 13⁄4 disease # ^

将 ST细胞以每孔 1. 5 X 10⁴细胞的细胞密度培养 16-24小时，再分别处理：猪融合重组型干扰素 (P IFNal-Fc 4a迎及未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal组） 16-24 小时，再以伪狂犬病毒 (Pseudorabies, PR病毒）（1 TCID₅。)接种该细胞，经 4-5天后，以 MTT法判读活性。 ST cells were cultured for 16-24 hours at a cell density of 1.5×10 ⁴ cells per well, and treated separately: porcine fusion recombinant interferon (P IFNal-Fc 4a incubated with porcine immunoglobulin IgG-Fc fragment The recombinant interferon (P IFNal group) was fused for 16-24 hours, and then the cells were inoculated with pseudorabies virus (Pseudorabies, PR virus) (1 TCID ₅ ). After 4-5 days, the activity was judged by MTT assay.

如表六及图 4所示，结果显示，相较于未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal Μ) , 本发明的猪融合重组型干扰素 (P IFNal-Fc 4a迎的抗 PR病毒活性较高。表六猪融合重组型干扰素 (P IFNal-Fc 4a组)及未与猪免疫球蛋白 IgG-Fc片段融合重组的干扰素 (P IFNal Μ)的抗 PR活性比较

相同地，将实施例四所得之含有其它猪融合重组型干扰素 (P IFNa-Fc) DNA序 7、 56、 58、 60、 62、 64、 66、 68、 70、 72、 74、 76、 78、 80、 82、 84、 86、 88、 90、 92、 94、 96、 98、 100、 102、 104、 106、 108、 110、 112、 114、 116、 118、 120、 122、 124、 126、 128、 130、 132、 134、 136、 138、 140、 142、 144、 146、 148、 150、 152、 154、 156、 158、 160、 162、 164、 166、 168、 170、 172、 174、 176、 178、 180、 182、 184、 186、 188、 190、 192、 194、 196、 198、 200、 202、 204、 206、 208、 210、 212、 214、 216、 218、 220及 222)的 _PSecTag2 (B) -IF a-Fc质体依照实施例五腿之方法转染至 CHO细胞株并表达该猪融合重组型干扰素 (P IFNa-Fc) (SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、 69、 71、 73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、 101、 103、 105、 107、 109、 111、 113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、 143、 145、 147、 149、 151、 153、 155、 157、 159、 161、 163、 165、 167、 169、 171、 173、 175、 177、 179、 181、 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、 211、 213、 215、 217、 219、 221及 223)，进而以实施例七、八戶; f¾ 方式分析各猪融合重组型干扰素 (P IFNa-Fc)抗 PRRS、 PR病毒的活性，结果发现，各猪融合重组型干扰素 (P IFNa-Fc)皆具有抗病毒活性，且皆车妹与动物免疫球蛋白 IgG-Fc片段融合重组之干扰素的抗病毒活性高。 As shown in Table 6 and Figure 4, the results show that the porcine fusion recombinant interferon (P IFNal-Fc) of the present invention is compared to the interferon (P IFNal Μ) which has not been fused with the porcine immunoglobulin IgG-Fc fragment. 4a is more active against PR virus. Table 6: Anti-PR of porcine fusion recombinant interferon (P IFNal-Fc 4a group) and interferon (P IFNal Μ) not fused with porcine immunoglobulin IgG-Fc fragment Activity comparison

Similarly, the other porcine fusion recombinant interferon (P IFNa-Fc) DNA sequence obtained in Example 4 was 7, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 , 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 , 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 _P SecTag2 of 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 and 222) - IF a-Fc plastid was transfected into CHO cell line according to the method of Example 5, and the porcine fusion recombinant interferon (P IFNa-Fc) was expressed (SEQ ID Nos: 8, 57, 59, 61, 63) , 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113 , 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221 and 223), and further analysis of the activity of anti-PRRS and PR viruses of each pig fusion recombinant interferon (P IFNa-Fc) by the method of Example 7, eight households; f3⁄4, and found that each pig fusion recombinant type Interferon (P IFNa-Fc) has antiviral activity, and the interferon which is fused with the animal immunoglobulin IgG-Fc fragment has high antiviral activity.

由上述实施例可知，本发明所提供的动物融合重组型干扰素在抗 DNA病毒以及抗 R A病毒活性上，皆较未与动物免疫球蛋白 IgG-Fc片段融合重组的干扰素的抗病毒活性高出许多。 It can be seen from the above examples that the animal fusion recombinant interferon provided by the present invention has higher antiviral activity against the DNA virus and the anti-RA virus than the interferon which has not been fused with the animal immunoglobulin IgG-Fc fragment. A lot.

上列详细说明系针对本发明之一可行实施例的具体说明，惟该实施例并非用以限制本发明的专利范围，脱离本发明技艺精神所为的等效实施更，均应包含于本案的专利范围中。 The detailed description above is a detailed description of a possible embodiment of the present invention, but the embodiment is not intended to limit the scope of the invention, and the equivalent implementation of the present invention should be included in the present invention. In the scope of patents.

Claims

权利要求书 Claim

1.一种动物融合重组型干扰素，包含一动物干扰素以及一动物免疫球蛋白 Fc片段。 An animal fusion recombinant interferon comprising an animal interferon and an animal immunoglobulin Fc fragment.

2.如权利要求 1腿之动物融合重组型干扰素，其特征在于，该动物干扰素以及该动物免疫球蛋白 Fc片段是由一连接子所连接。 The animal fusion recombinant interferon according to claim 1, wherein the animal interferon and the animal immunoglobulin Fc fragment are linked by a linker.

3.如权利要求 1或2臓之动物融合重组型干扰素，其特征在于，该动物干扰素具有一難酸序列，该¼¾酉辨列选自于由 SEQ ID Nos : 2、 17、 19、 21、 23、 25、 27、 29、 31、 33、 35、 37、 39、 41、 43、 45及 47所 j¾之群组中至少一者。 The animal fusion recombinant interferon according to claim 1 or 2, wherein the animal interferon has a difficult acid sequence selected from SEQ ID Nos: 2, 17, 19, 21 At least one of the groups of 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, and 47 j3⁄4.

4.如权利要求 1或2臓之动物融合重组型干扰素，其特征在于，该动物干扰素具有一難酸序列，该¼¾酉辨列与选自于由 SEQ ID Nos : 2、 17、 19、 21、 23、 25、 27、 29、 31、 33、 35、 The animal fusion recombinant interferon according to claim 1 or 2, wherein the animal interferon has a difficult acid sequence, and the sequence is selected from SEQ ID Nos: 2, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,

37、 39、 41、 43、 45及 47所賊之群 S少一者的序列具有至少 80%序列同源性。 Groups of 37, 39, 41, 43, 45, and 47 thieves have a sequence of at least 80% sequence homology.

5.如权利要求 1或 2 动物融合重组型干扰素，其特征在于，该动物免疫球蛋白 Fc片段具有一 5@酉辨列，该氨基酉辨列选自于由 SEQ ID Nos : 4、 49、 51、 53、及 55所賊之群组中至少一者。 The animal fusion recombinant interferon according to claim 1 or 2, wherein the animal immunoglobulin Fc fragment has a 5@酉 discrimination column selected from SEQ ID Nos: 4, 49 At least one of the groups of 51, 53 and 55 thieves.

6.如权利要求 1或 2 动物融合重组型干扰素，其特征在于，该动物免疫球蛋白 Fc片段具有一 S翻亨列，该氨基酸序列与选自于由 SEQ ID Nos : 4、 49、 51、 53、及 55所组成之群组至少一者的序列具有至少 80%序列同源性。 6. The animal fusion recombinant interferon according to claim 1 or 2, wherein the animal immunoglobulin Fc fragment has a S-replacement sequence selected from SEQ ID Nos: 4, 49, 51 The sequence of at least one of the groups consisting of 53, 53 and 55 has at least 80% sequence homology.

7.如权利要求 2戶； ¾¾¾物融合重组型干扰素，其特征在于，该连接子具有如 SEQ ID No: 6 所示之序列。 The recombinant interferon according to claim 2, wherein the linker has a sequence as shown in SEQ ID No: 6.

8.如权利要求 2腿之动物融合重组型干扰素，其特征在于，该连接子具有一讓顯，该難辦列与 SEQ ID No: 6所示之序列具有至少 80%序列同源性。 The animal fusion recombinant interferon according to claim 2, wherein the linker has a phenotype which has at least 80% sequence homology with the sequence of SEQ ID No: 6.

9.如权利要求 2臓之动物融合重组型干扰素，其特征在于，该动物融合重组型干扰素具有一¼¾酉辨列，该氨基酉辨列选自于由 SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、 69、 71、 73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、 101、 103、 105、 107、 109、 111、 113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、 143、 145、 147、 149、 151、 153、 155、 157、 159、 161、 163、 165、 167、 169、 171、 173、 175、 177、 179、 181、 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、 211、 213、 215、 217、 219、 221及 223腿成之群组。 The animal fusion recombinant interferon according to claim 2, wherein the animal fusion recombinant interferon has a 1⁄4⁄4酉 list, and the amino group is selected from SEQ ID Nos: 8, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, Groups of 209, 211, 213, 215, 217, 219, 221, and 223 legs.

10.如权利要求 2臓之动物融合重组型干扰素，其特征在于，该动物融合重组型干扰素具有一„ 列，该氨基酸序列与选自于由 SEQ ID Nos : 8、 57、 59、 61、 63、 65、 67、 69、 71、 10. The animal fusion recombinant interferon according to claim 2, wherein the animal fusion recombinant interferon has a sequence selected from SEQ ID Nos: 8, 57, 59, 61 , 63, 65, 67, 69, 71,

73、 75、 77、 79、 81、 83、 85、 87、 89、 91、 93、 95、 97、 99、皿、 103、 105、 107、 109、 m、73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, Dish, 103, 105, 107, 109, m,

113、 115、 117、 119、 121、 123、 125、 127、 129、 131、 133、 135、 137、 139、 141、 143、 145、113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,

147、 149、 151、 153、 155、 157、 159、 161、 163、 165、 167、 169、 m、 173、 175、 177、 179、147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, m, 173, 175, 177, 179,

181、 183、 185、 187、 189、 191、 193、 195、 197、 199、 201、 203、 205、 207、 209、 211、 213、 215、 217、 219、 221及 223所賊之群组之一的序列具有至少 80%序列同源性。 One of the groups of thieves 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, and 223 The sequence has at least 80% sequence homology.

11.一种编码如权利要求 1腿之动物融合重组型干扰素的多核苷酸。 11. A polynucleotide encoding an animal fusion recombinant interferon as claimed in claim 1.

12.一种制备一动物融合重组型干扰素的方法，包含将带有如权利要求 11腿之多核苷酸的哺乳细胞以含有血清的培养基培养至该细赃长稳定后，更换为青培养基继離养，并于每隔 1至 5天更新的 ¾1清培养基以及收获该动物融合重组型干扰素。 A method for producing an animal fusion recombinant interferon, comprising: culturing a mammalian cell carrying the polynucleotide of the leg of claim 11 in a serum-containing medium until the fine sputum is stable, and replacing it with a qing medium Following the colonization, the 3⁄41 clear medium was updated every 1 to 5 days and the animal fusion recombinant interferon was harvested.

13.如权利要求 12戶； f¾ 方法，其中该含有血清的培养基的血清^ *为 0. 1至 1(H林口、百分比。 13. The method according to claim 12, wherein the serum of the serum-containing medium is from 0.1 to 1 (H, mouth, percentage).

14.一种如权利要求 1戶; M之动物融合重组型干扰素在制备动物抗病毒药物中的用途。 14. Use of an animal fusion recombinant interferon according to claim 1 for the preparation of an animal antiviral drug.