TWI638827B - Recombinant antigen with cross protection and animal vaccine composition including the same - Google Patents
Recombinant antigen with cross protection and animal vaccine composition including the same Download PDFInfo
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Abstract
本發明有關於一種交叉保護性重組抗原以及含有此交叉保護性重組抗原之疫苗組成物。前述之交叉保護性重組抗原包括為豬丹毒桿菌之表面保護抗原N端片段(rSpaA-N)之重組蛋白，而前述疫苗組成物。由此製得動物用疫苗組成物，以誘發至少一種受免疫之對象的體內產生抗體，同時刺激細胞性及體液性免疫反應，以保護受免疫之對象免於至少二不同血清型之豬丹毒桿菌之感染。 The present invention relates to a cross-protective recombinant antigen and a vaccine composition comprising the cross-protective recombinant antigen. The aforementioned cross-protective recombinant antigen includes a recombinant protein which is a surface protective antigen N-terminal fragment of porcine typhimurium (rSpaA-N), and the aforementioned vaccine composition. Thus, an animal vaccine composition is prepared to induce the production of antibodies in at least one immunized subject, while stimulating cellular and humoral immune responses to protect the immunized subject from at least two different serotypes of E. sinensis Infection.
Description
本發明是有關於一種重組抗原及含彼之疫苗組成物，特別是有關於一種交叉保護性重組抗原及含彼之動物疫苗組成物。 The present invention relates to a recombinant antigen and a vaccine composition therefor, and more particularly to a cross-protective recombinant antigen and a vaccine composition containing the same.
豬丹毒(Swine erysipelas；SE)是由豬丹毒桿菌(Erysipelothrix rhusiopathiae,E.r)引起的一種急性熱性傳染病，其主要病徵為高熱、急性敗血症、皮膚疹塊(亞急性)、慢性疣狀心內膜炎、皮膚壞死以及多發性非化膿性關節炎。豬丹毒桿菌常存於30%至50%的健康豬隻之扁桃腺及淋巴組織中，藉由傷口、糞便及口鼻分泌物傳染，擴大疫情傳播，且豬丹毒桿菌常感染於肥育期的豬隻以及懷孕母豬，故造成養豬業者的鉅大經濟損失。 Swine erysipelas (SE) is an acute heat-borne disease caused by Erysipelothrhr rhusiopathiae ( Er ). Its main symptoms are hyperthermia, acute sepsis, skin rash (subacute), chronic sacral endocardium. Inflammation, skin necrosis, and multiple non-suppurative arthritis. E. sinensis is often found in the tonsils and lymphoid tissues of healthy pigs from 30% to 50%. It spreads through wounds, feces and oral and nasal secretions, and spreads the epidemic. Pigs are often infected in the finishing period. Only the pregnant sows cause huge economic losses for the pig farmers.
過去研究顯示，豬丹毒桿菌的致病機轉可能為當豬丹毒桿菌感染宿主時，因豬丹毒桿菌之外層具有莢膜(capsular)保護，可免於免疫細胞吞噬消化，形成抗吞噬防禦機制，因而可長存於宿主體內並繁殖，豬丹毒桿菌再藉由 釋放神經氨酸酶(neuraminidase)破壞宿主內皮細胞表面結構，有利於使更多豬丹毒桿菌以自身表面保護蛋白Spa黏附於宿主內皮細胞上，進而入侵宿主微血管，使豬丹毒桿菌進入宿主血液循環系統，導致感染惡化，甚至引發敗血症。 Past studies have shown that the pathogenesis of E. typhimurium may be when the E. sinensis infection host, because the outer layer of E. sinensis has capsular protection, can avoid immune cell phagocytosis and digestion, forming an anti-phagocytic defense mechanism, Therefore, it can persist in the host and multiply, and the bacterium The release of neuraminidase destroys the surface structure of host endothelial cells, which helps to make more porcine dystrophies adhere to the host endothelial cells with their own surface protective protein Spa, thereby invading the host microvessels, allowing the porcine bacillus to enter the host blood circulation system. , leading to worsening infections and even causing sepsis.
過去研究結果顯示，以豬丹毒桿菌表面保護抗原(SpaA)的完整多肽序列製成單株抗體(I2A)，證實能夠辨識16種血清型，亦能使豬丹毒桿菌緊密地黏附於細胞。 Past studies have shown that a single antibody (I2A) is produced from the complete polypeptide sequence of the A. faecalis surface-protecting antigen (SpaA), which confirms that 16 serotypes can be identified, and also enables the typhoid bacillus to adhere tightly to cells.
另外，現今市售疫苗產品之抗原對於不同血清型的豬丹毒桿菌的交叉保護力有限，無法引起長效型的交叉免疫保護，其效能亦備受質疑。 In addition, the antigens of commercially available vaccine products have limited cross-protection against different serotypes of E. sinensis, and cannot cause long-acting cross-immunization protection, and their efficacy is also questioned.
有鑑於此，亟需發展一種新穎、有效且適用於動物用之交叉保護抗原重組蛋白，以改善習知市售疫苗的缺點。 In view of this, there is an urgent need to develop a novel, effective and suitable cross-protective antigen recombinant protein for use in animals to improve the disadvantages of conventional commercially available vaccines.
因此，本發明之一態樣是在提供一種單離之核酸，其包含如序列辨識編號(SEQ ID NO.)：1所示之核酸序列。 Accordingly, one aspect of the invention provides an isolated nucleic acid comprising a nucleic acid sequence as set forth in SEQ ID NO.: 1.
本發明之另一態樣係在提供一種重組載體，其包含如SEQ ID NO.：1所示之核酸序列。 Another aspect of the invention provides a recombinant vector comprising the nucleic acid sequence set forth in SEQ ID NO.: 1.
本發明之又一態樣係在提供一種交叉保護性重組抗原，其包含如SEQ ID NO.：1所示之核酸序列經原核生物表現系統表現之胺基酸序列。 A further aspect of the invention provides a cross-protective recombinant antigen comprising an amino acid sequence as represented by the prokaryotic expression system of the nucleic acid sequence set forth in SEQ ID NO.: 1.
本發明之再一態樣係在提供一種抗豬丹毒桿菌 感染之疫苗組成物，其包含上述之交叉保護性重組抗原，以提供受免疫之動物對不同血清型的豬丹毒桿菌產生交叉保護力。 A further aspect of the present invention provides an anti-Rhamnoid An infectious vaccine composition comprising the above cross-protective recombinant antigen to provide cross-protection of the immunized animal against different serotypes of E. rhus.
根據本發明之上述態樣，提出一種單離之核酸，其包含如序列辨識編號(SEQ ID NO.)：1所示之核酸序列。 According to the above aspect of the present invention, there is provided an isolated nucleic acid comprising the nucleic acid sequence as shown in the sequence identification number (SEQ ID NO.): 1.
根據本發明之另一態樣，提出一種重組載體，其包含如SEQ ID NO.：1所示之核酸序列。 According to another aspect of the invention, a recombinant vector comprising the nucleic acid sequence set forth in SEQ ID NO.: 1 is provided.
根據本發明之又一態樣，提出一種交叉保護性重組抗原，其包含如SEQ ID NO.：1所示之一核酸序列經原核生物表現系統表現之胺基酸序列。 According to still another aspect of the present invention, a cross-protective recombinant antigen comprising an amino acid sequence represented by a prokaryotic expression system of a nucleic acid sequence set forth in SEQ ID NO.: 1 is provided.
根據本發明之再一態樣，提出一種抗豬丹毒桿菌感染之疫苗組成物，其包如SEQ ID NO.：1所示之一核酸序列經原核生物表現系統表現之交叉保護性重組抗原及醫藥學上可接受之載劑。 According to still another aspect of the present invention, a vaccine composition for infection against E. sinensis comprising a cross-protective recombinant antigen and a pharmaceutical agent represented by a prokaryotic expression system of a nucleic acid sequence represented by SEQ ID NO. A school-acceptable carrier.
依據本發明一實施例，上述交叉保護性重組抗原可例如為豬丹毒桿菌表面保護抗原N端片段(rSpaA-N)之重組蛋白。 According to an embodiment of the present invention, the cross-protective recombinant antigen may be, for example, a recombinant protein of an N-terminal fragment of a porcine typhimurium surface protective antigen (rSpaA-N).
依據本發明一實施例，上述抗豬丹毒桿菌感染之疫苗組成物可包含但不限於每毫升100微克(μg)至400μg之交叉保護性重組抗原。在一例示中，上述抗豬丹毒桿菌感染之疫苗組成物可包含但不限於每毫升100μg至200μg之交叉保護性重組抗原。 According to an embodiment of the present invention, the vaccine composition for anti-P. sinensis infection may include, but is not limited to, 100 micrograms (μg) to 400 μg of cross-protective recombinant antigen per ml. In an exemplary embodiment, the vaccine composition for anti-P. sinensis infection may include, but is not limited to, from 100 μg to 200 μg of cross-protective recombinant antigen per ml.
依據本發明一實施例，上述醫藥學上可接受之 載劑可包含但不限於佐劑及/或免疫增效劑。 According to an embodiment of the invention, the above pharmaceutically acceptable Carriers can include, but are not limited to, adjuvants and/or immunopotentiators.
依據本發明一實施例，上述抗豬丹毒桿菌感染之疫苗組成物可經由例如口服途徑、非口服途徑或噴霧吸入途徑投予一對象，以同時保護該對象免於至少二不同血清型之豬丹毒桿菌之感染。在一例示中，前述非口服途徑可包含但不限於肌肉注射途徑、皮下注射途徑、皮內注射途徑或腹腔注射途徑。 According to an embodiment of the present invention, the vaccine composition for the infection of E. sinensis can be administered to a subject via, for example, an oral route, a non-oral route or a spray inhalation route to simultaneously protect the subject from at least two different serotypes of porcine erysipelas. Bacillus infection. In one example, the aforementioned non-oral route can include, but is not limited to, an intramuscular route, a subcutaneous route, an intradermal route, or an intraperitoneal route.
依據本發明一實施例，上述對象為豬。 According to an embodiment of the invention, the object is a pig.
依據本發明一實施例，上述豬丹毒桿菌之至少二不同血清型可包括但不限於血清型第1a型以及血清型第2型。應用本發明之交叉保護性重組抗原，其係利用豬丹毒桿菌表面保護抗原N端片段(rSpaA-N)之重組蛋白作為抗原，以製得動物用疫苗組成物，以誘發至少一種受免疫動物之體內產生抗體，同時刺激細胞性及體液性免疫反應，以保護受免疫之對象免於至少二不同血清型之豬丹毒桿菌之感染。 According to an embodiment of the present invention, at least two different serotypes of the porcine typhoid bacteria may include, but are not limited to, serotype type 1a and serotype type 2. The cross-protective recombinant antigen of the present invention is obtained by using a recombinant protein of a N-terminal fragment of a porcine plague surface-protecting antigen (rSpaA-N) as an antigen to prepare an animal vaccine composition to induce at least one animal to be immunized The production of antibodies in vivo stimulates both cellular and humoral immune responses to protect the immunized subject from infection by at least two different serotypes of E. rhus.
401/403/405/501/503/505/601/603/605/701/705‧‧‧直條 401/403/405/501/503/505/601/603/605/701/705‧‧ ‧ straight bars
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之詳細說明如下： The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.
【圖1】係顯示根據本發明一實施例利用特異性引子對所得之交叉保護性重組抗原核酸序列的瓊脂膠體電泳照片。 Fig. 1 is a photograph showing an agar colloidal electrophoresis of a cross-protective recombinant antigen nucleic acid sequence obtained by using a specific primer pair according to an embodiment of the present invention.
【圖2】係顯示根據本發明一實施例之交叉保護性重 組抗原的蛋白質電泳膠體照片。 2 shows a cross-protective weight according to an embodiment of the present invention. A photograph of a protein electrophoresis gel of a group of antigens.
【圖3】係顯示根據本發明一實施例之交叉保護性重組抗原的西方墨點法偵測照片。 Fig. 3 is a Western blot detection photograph showing a cross-protective recombinant antigen according to an embodiment of the present invention.
【圖4】係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG之抗體力價直條圖。 Fig. 4 is a graph showing the antibody valence of IgG in serum after inoculation of a vaccine composition against porcine typhoon infection of the first embodiment of the mouse according to an embodiment of the present invention.
【圖5】係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG2a之抗體力價直條圖。 Fig. 5 is a graph showing the antibody valence of IgG2a in the serum after inoculation of the vaccine composition of the anti-pigmatovirus infection of the first embodiment of the mouse according to an embodiment of the present invention.
【圖6】係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG1之抗體力價直條圖。 Fig. 6 is a graph showing the antibody valence of IgG1 in the serum after inoculation of the vaccine composition of the mouse against the porcine bacillus infection of Example 1 according to an embodiment of the present invention.
【圖7】係繪示根據本發明一實施例之豬隻利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG之抗體力價直條圖。 Fig. 7 is a bar graph showing the antibody titer of IgG in the serum after inoculation of the vaccine composition of the pig anti-P. sinensis infection of the first embodiment according to an embodiment of the present invention.
本說明書中所述之所有技術性及科學術語，除非另外有所定義，皆為該所屬領域具有通常技藝者可共同瞭解的意義。 All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.
承前所述，本發明提供一種交叉保護性重組抗原，其包含豬丹毒桿菌表面保護抗原(SpaA)的N端序列之重組蛋白，可應用於動物疫苗組成物。 As described above, the present invention provides a cross-protective recombinant antigen comprising a recombinant protein of an N-terminal sequence of a porcine typhimurium surface protective antigen (SpaA), which can be applied to an animal vaccine composition.
豬丹毒桿菌為革蘭氏陽性短桿菌，依肽聚醣可 分為23種血清型，其中以血清型第一型與第二型較為常見。主要存在於豬隻扁桃腺及淋巴組織，再經由口鼻分泌物、皮膚傷口及糞便之平行傳染，為人畜共通傳染病。過去研究指出將豬丹毒桿菌表面保護抗原A免疫於兔子，其血清中IgG抗體具有能辨識16種不同血清型，顯示SpaA可能具有交叉保護豬丹毒.之不同血清型的潛力。 E. sinensis is a Gram-positive Brevibacterium, which can be peptidoglycan Divided into 23 serotypes, of which serotypes 1 and 2 are more common. It mainly exists in the tonsils and lymphoid tissues of pigs, and is transmitted through parallel infections of oral and nasal secretions, skin wounds and feces, and is a common infectious disease for humans and animals. Past studies have indicated that immunization of Phytophthora infestans surface-protected antigen A in rabbits with IgG antibodies in serum can identify 16 different serotypes, suggesting that SpaA may have the potential to cross-protect different serotypes of porcine erysipelas.
在一實施例中，上述之交叉保護性重組抗原可例如由含有一核酸序列的重組載體所表現之胺基酸序列。在一例示中，上述核酸序列可例如為如序列辨識編號(SEQ ID NO.)：1所示單離的核酸。 In one embodiment, the cross-protective recombinant antigen described above can be, for example, an amino acid sequence represented by a recombinant vector comprising a nucleic acid sequence. In an exemplary embodiment, the nucleic acid sequence can be, for example, an isolated nucleic acid as shown in Sequence Identification Number (SEQ ID NO.): 1.
申言之，上述核酸序列可例如為豬丹毒桿菌之表面保護抗原基因的N端片段(rSpaA-N)，其由如SEQ ID NO：2所示之上游引子及SEQ ID NO：3所示之下游引子由豬丹毒桿菌之基因組DNA增幅出的核酸片段。前述使用之豬丹毒桿菌是由國立屏東科技大學動物疫苗科技研究所朱純燕教授所提供，屬於血清型第1a型。 In the above, the nucleic acid sequence may be, for example, an N-terminal fragment (rSpaA-N) of the surface protective antigen gene of E. sinensis, which is represented by the upstream primer shown in SEQ ID NO: 2 and SEQ ID NO: 3. The downstream primer is a nucleic acid fragment amplified by the genomic DNA of A. faecalis. The aforementioned porcine bacillus is provided by Professor Zhu Chunyan of the National Institute of Animal Vaccine Science and Technology of Pingtung University of Science and Technology, and belongs to serotype 1a.
上述rSpaA-N基因序列為1252bp，該蛋白產物大小約為69KDa。在先期測試結果中，利用感染後豬隻血清分析，證實rSpaA-N重組蛋白仍保有免疫原性，但分子較小、生物安全性較高又易於量產，可作為豬丹毒桿菌重組次單位疫苗之抗原蛋白用。 The above rSpaA-N gene sequence is 1252 bp, and the protein product size is about 69 KDa. In the preliminary test results, using the serum analysis of infected pigs, it was confirmed that rSpaA-N recombinant protein still retains immunogenicity, but the molecule is small, biosafety and easy to mass production, and can be used as recombinant subunit vaccine of E. sinensis. For antigenic proteins.
在一實施例中，前述rSpaA-N重組蛋白可搭配雙相油質佐劑，用於製備豬丹毒桿菌重組次單位疫苗，並進一步免疫於動物體內，以同時誘導細胞性及體液性免疫反 應，更同時具有保護交叉不同血清型之豬丹毒桿菌感染。 In one embodiment, the rSpaA-N recombinant protein can be used in combination with a biphasic oil adjuvant for preparing a recombinant subunit vaccine of E. sinensis, and further immunizing the animal to simultaneously induce cellular and humoral immunity. It should, at the same time, protect against infection with E. coli that crosses different serotypes.
另外，在其他實施例中，本發明含有rSpaA-N重組蛋白之疫苗組成物於受免疫動物之血清抗體結果證實，其血清中IgG、IgG2a、IgG1等抗體較市售疫苗組及控制組(未進行免疫)皆具顯著性差異，可發展為次單位疫苗之抗原。 In addition, in other embodiments, the vaccine composition containing the rSpaA-N recombinant protein of the present invention is confirmed by serum antibody results of the immunized animal, and the antibodies such as IgG, IgG2a, and IgG1 in the serum are compared with the commercially available vaccine group and the control group (not Immunization) has significant differences and can be developed as an antigen for subunit vaccines.
本發明此處所稱之「佐劑」係指可增加動物對此疫苗產生免疫反應的物質，其可與疫苗在相同時間及相同位置上被給與，或者在不同時間給與，例如作為加強劑。佐劑亦可被有利地以同一種方法或不同於給與疫苗之方法或位置來給與受免疫之對象。 The term "adjuvant" as used herein refers to a substance which increases the immune response of an animal to the vaccine, which can be administered at the same time and at the same position as the vaccine, or at different times, for example as a reinforcing agent. . The adjuvant may also be advantageously administered to the subject to be immunized in the same manner or differently from the method or location in which the vaccine is administered.
在一實施例中，上述佐劑可包含但不限於油質佐劑、水質佐劑(如：氫氧化鋁)、雙相油質佐劑(如：脂質體(Liposome)、水包油包水(W/O/W)型)、生物型佐劑(如：細胞激素、CpG寡核苷酸、細菌類毒素toxoid)等。 In one embodiment, the above adjuvant may include, but is not limited to, an oil adjuvant, a water adjuvant (eg, aluminum hydroxide), a dual phase oil adjuvant (eg, liposome, water-in-oil water-in-water) (W/O/W) type), biotype adjuvant (such as: cytokines, CpG oligonucleotides, bacterial toxoid).
上述雙相油質佐劑可進一步參考習知方法製備，或參考例如本案發明人之一朱純燕等人於2007年在臺灣獸醫學雜誌第33期第206-211頁發表之題目為「水包油包水(W/O/W)油質佐劑對牛流行熱疫苗抗體消長之影響」一文所揭示的方法製備，在此一併列為參考文獻。 The above-mentioned two-phase oily adjuvant can be further prepared by referring to a conventional method, or referenced, for example, to one of the inventors of the present invention, Zhu Chunyan et al., 2007, Taiwan Journal of Veterinary Medicine, No. 33, pp. 206-211, entitled "Water-in-water oil". The preparation of the water-in-water (W/O/W) oily adjuvant on the growth and decline of antibodies to bovine epidemic fever vaccines is prepared by reference herein.
在一實施例中，上述疫苗組成物可經由例如口服途徑、非口服途徑或噴霧吸入途徑投予一對象，端視實際需求而定。在一例示中，前述非口服途徑可包含但不限於肌肉注射途徑、皮下注射途徑、皮內注射途徑或腹腔注射途 徑。在其他例示中，前述受免疫之對象可例如為一經濟動物，其中經濟動物可包括但不限於家畜，例如豬、牛或羊之家畜。 In one embodiment, the vaccine composition described above can be administered to a subject via, for example, an oral route, a non-oral route, or a spray inhalation route, depending on actual needs. In one example, the aforementioned non-oral route may include, but is not limited to, an intramuscular route, a subcutaneous route, an intradermal route, or an intraperitoneal route. path. In other embodiments, the aforementioned immunized subject can be, for example, an economic animal, wherein the economic animal can include, but is not limited to, livestock, such as livestock of pigs, cattle, or sheep.
在應用於動物疫苗組成物時，此疫苗組成物可包含但不限於抗原以及生物型佐劑，其中疫苗組成物可包含例如200μg/mL之生物型佐劑。在一實施例中，上述疫苗組成物可對每頭豬隻(三至四週齡，體重約4公斤至6公斤)施予400微克(μg)之生物型佐劑。在一實施例中，上述疫苗組成物可包含但不限於抗原為豬巴氏桿菌類毒素蛋白(PMT)。有關豬隻與小鼠之疫苗組成物接種劑量的換算，係根據Hsueh K.-J.等人於2014年在期刊Transboundary and Emerging Diseases第61期第6卷第e35-43頁發表、其標題為「Evaluation on a Streptococcus suis Vaccine Using Recombinant sSao-L Protein Manufactured by Bioreactors as the Antigen in Pigs」一文進行換算，其全文在此一併列為本發明之參考文獻。 When applied to an animal vaccine composition, the vaccine composition can include, but is not limited to, an antigen as well as a biologic adjuvant, wherein the vaccine composition can comprise, for example, a biosupplier of 200 [mu]g/mL. In one embodiment, the vaccine composition described above is capable of administering 400 micrograms (μg) of a biologic adjuvant to each pig (three to four weeks old, weighing about 4 to 6 kilograms). In one embodiment, the vaccine composition described above may include, but is not limited to, an antigen that is a Pasteurella toxoid toxin protein (PMT). The conversion of vaccine doses for pigs and mice is based on Hsueh K.-J. et al., 2014, Journal of Transboundary and Emerging Diseases, Vol. 6, Vol. 6, pp. e35-43, entitled The "Evaluation on a Streptococcus suis Vaccine Using Recombinant sSao-L Protein Manufactured by Bioreactors as the Antigen in Pigs" is hereby incorporated by reference in its entirety.
本發明以原核表現系統表現之rSpaA-N重組蛋白作為交叉保護性重組抗原，並以小鼠試驗評估含有rSpaA-N重組蛋白之疫苗組成物的保護效力以及有效劑量，再與W/O/W型雙相油質乳劑搭配試製成重組次單位疫苗，以小鼠試驗證實rSpaA-N重組蛋白具有保護受免疫之對象免於至少二不同血清型之豬丹毒桿菌之感染之功效。 The invention uses the rSpaA-N recombinant protein expressed by the prokaryotic expression system as a cross-protective recombinant antigen, and evaluates the protective efficacy and effective dose of the vaccine composition containing the rSpaA-N recombinant protein in a mouse test, and then works with W/O/W. The two-phase oil emulsion was tested to prepare a recombinant subunit vaccine, and the mouse test confirmed that the rSpaA-N recombinant protein has the effect of protecting the infected subject from infection by at least two different serotypes of E. sinensis.
以下利用數個實施例以說明本發明之應用，然其並非用以限定本發明，本發明技術領域中具有通常知識 者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾。 The following examples are used to illustrate the application of the present invention, but are not intended to limit the present invention, and have general knowledge in the technical field of the present invention. Various changes and modifications can be made without departing from the spirit and scope of the invention.
此實施例使用之豬丹毒桿菌是由國立屏東科技大學動物疫苗科技研究所朱純燕教授所提供，屬於血清型第1a型。 The erysipemus bacterium used in this embodiment is provided by Professor Zhu Chunyan of the National Institute of Animal Vaccine Science and Technology of Pingtung University of Science and Technology, and belongs to serotype 1a.
將豬丹毒桿菌培養於含5%之脫纖維羊血之血液瓊脂培養基上，於37℃恆溫培養箱中培養48小時後，挑選單一菌落接種於3mL之腦心浸液(Brain Heart Infusion，BHI；BD,MD,USA)培養液中，內含5%之雞血清(chicken serum；CS)，以37℃震盪培養48h，最後將菌液置於4℃備用。 The porcine bacillus was cultured on a blood agar medium containing 5% defibrinated sheep blood, and cultured in a 37 ° C incubator for 48 hours, and then a single colony was selected and inoculated into 3 mL Brain Heart Infusion (BHI; BD, MD, USA) medium containing 5% chicken serum (CS), cultured at 37 ° C for 48 h, and finally placed at 4 ° C for use.
1. 分離基因組DNAIsolation of genomic DNA
此實施例利用市售DNA萃取套組，例如Blood & Tissue Genomic DNA Extraction Miniprep System(Viogene,CA,USA)，萃取上述菌液的基因組DNA。根據製造商的使用手冊操作，即可萃取基因組DNA，並置於-20℃備用。 This example uses a commercially available DNA extraction kit, such as the Blood & Tissue Genomic DNA Extraction Miniprep System (Viogene, CA, USA), to extract the genomic DNA of the bacterial solution. Genomic DNA can be extracted and stored at -20 °C for use according to the manufacturer's instruction manual.
2. 增幅rSpaA-N核酸片段2. Amplifying the rSpaA-N nucleic acid fragment
此實施例使用之引子對是針對豬丹毒桿菌SpaA-N基因，並經IEDB(Immune Epitope database) 軟體分析序列親/疏水性(http：//tools.immuneepitope.org/tools/bcell/iedb_input)者，可例如SEQ ID NO：2所示之正向引子以及如SEQ ID NO：3所示之反向引子，以以95℃ 1分鐘、57℃ 1分鐘及72℃ 2分鐘為一個循環進行PCR，共進行30個循環，藉此增幅出PCR產物，其中正向引子具有限制酶BamH I的切位序列，反向引子具有限制酶Hind III的切位序列，而PCR產物為用於表現豬丹毒桿菌之表面保護抗原N端片段(rSpaA-N)之重組蛋白的核酸片段。 The primer pair used in this example is directed against the A. faecalis SpaA-N gene and is IEDB (Immune Epitope database). The software analysis sequence affinity/hydrophobicity (http://tools.immuneepitope.org/tools/bcell/iedb_input) can be, for example, the forward primer shown in SEQ ID NO: 2 and the reverse as shown in SEQ ID NO: 3. To the primer, PCR was carried out in one cycle of 95 ° C for 1 minute, 57 ° C for 1 minute, and 72 ° C for 2 minutes for a total of 30 cycles, thereby increasing the PCR product, wherein the forward primer has a restriction enzyme BamH I cleavage position. The sequence, the reverse primer has a cleavage sequence of the restriction enzyme Hind III, and the PCR product is a nucleic acid fragment of a recombinant protein for expressing the N-terminal fragment of the surface protective antigen of porcine typhimurium (rSpaA-N).
3. 確認rSpaA-N核酸片段3. Confirm rSpaA-N nucleic acid fragment
上述PCR產物利用0.8~1.2%(W/V)之洋菜膠體，於TAE緩衝溶液[Tris-acetate-EDTA(TAE)緩衝溶液，含40mM Tris-HCL、20mM glacial acid以及1mM EDTA，pH 8.0]中，以電壓100伏特進行30至40分鐘電泳。之後，利用市售影像系統(例如UVIDOC影像系統；Uvitec,Cambridge,UK)，確認增幅之PCR產物(即rSpaA-N核酸片段)，其大小與預期1252bp相符，如圖1之箭頭所示。 The above PCR product utilizes 0.8-1.2% (w/V) of acacia colloid in TAE buffer solution [Tris-acetate-EDTA (TAE) buffer solution containing 40 mM Tris-HCL, 20 mM glacial acid and 1 mM EDTA, pH 8.0] The electrophoresis was carried out for 30 to 40 minutes at a voltage of 100 volts. Thereafter, the amplified PCR product (i.e., rSpaA-N nucleic acid fragment) was confirmed using a commercially available imaging system (e.g., UVIDOC imaging system; Uvitec, Cambridge, UK), the size of which corresponds to the expected 1252 bp, as indicated by the arrows in FIG.
4. 分離PCR反應產物4. Isolation of PCR reaction products
使用AxyPrep TM PCR Clean up Kit(Axygen,CA,USA)，根據製造商的使用手冊操作，即可由PCR反應產物之DNA得到純化的rSpaA-N核酸片段，經確認DNA序列無誤(如SEQ ID NO：1所示)後，置於-20℃備用。 The purified rSpaA-N nucleic acid fragment can be obtained from the DNA of the PCR reaction product using the AxyPrepTM PCR Clean up Kit (Axygen, CA, USA) according to the manufacturer's instruction manual, and the DNA sequence is confirmed to be correct (eg SEQ ID NO: After 1), put it at -20 °C for later use.
5. 製備pET-32a/rSpaA-N重組質體5. Preparation of recombinant plasmid pET-32a/rSpaA-N
將純化後之rSpaA-N核酸片段與pET-32a質體分別以相同的限制酶進行切割，再以T4 DNA連接酶(400U/μL)(TaKaRa,Shiga,Japan)於16℃進行4小時的接合反應，以獲pET-32a/SpaA-N重組質體。 The purified rSpaA-N nucleic acid fragment and the pET-32a plasmid were cleaved with the same restriction enzyme, respectively, and then ligated with T4 DNA ligase (400 U/μL) (TaKaRa, Shiga, Japan) at 16 ° C for 4 hours. The reaction was carried out to obtain a recombinant plasmid of pET-32a/SpaA-N.
1. 轉形作用Transformation effect
此實施例係進行轉形作用。首先，將10μL之pET-32a/rSpaA-N重組質體，加入100μL之E.coli BL21(DE3)勝任細胞中均勻混合，於42℃水浴中進行90秒之熱休克(heat shock)程序處理，以獲得rSpaA-N重組轉形株。然後，rSpaA-N重組轉形株利用含有ampicillin(100μg/mL)(MDBio,Taipei,Taiwan)之LB培養基，置於37℃恆溫培養箱中培養14至16小時。 This embodiment performs a transformation. First, 10 μL of pET-32a/rSpaA-N recombinant plasmid was uniformly mixed with 100 μL of E. coli BL21 (DE3) competent cells, and subjected to a heat shock program for 90 seconds in a 42 ° C water bath. The rSpaA-N recombinant transgenic strain was obtained. Then, the rSpaA-N recombinant transformant was cultured in an LB medium containing ampicillin (100 μg/mL) (MDBio, Taipei, Taiwan) and cultured in a 37 ° C incubator for 14 to 16 hours.
2. 分離重組轉形株的pET-32a/rSpaA-N重組質體DNA2. Isolation of recombinant plasmid pET-32a/rSpaA-N recombinant plastid DNA
將轉形成功之pET-32a/rSpaA-N的重組轉形株，利用Plasmid miniprep purification kit(GeneMark,Taichung,Taiwan)，根據製造商的使用手冊操作，即可萃取得到pET-32a/rSpaA-N重組質體DNA，並置於-20℃備用。 The recombinant transgenic strain of pET-32a/rSpaA-N, which was successfully transformed, was extracted using the Plasmid miniprep purification kit (GeneMark, Taichung, Taiwan) according to the manufacturer's manual, and pET-32a/rSpaA-N was extracted. Recombinant plastid DNA was reconstituted and placed at -20 °C until use.
3. 表現rSpaA-N重組蛋白3. Expression of rSpaA-N recombinant protein
挑選前述轉形成功且選殖表現之重組蛋白轉形 株(E.coli BL 21/rSpaA-N)的單一菌落，接種於3mL之LB(Difco,MD,USA；含濃度100μg/mL之ampicillin)培養液中，於37℃培養箱內進行振盪培養。當菌液的吸光值達到OD600nm 0.6至0.8時，加入isopropyl-β-D-thiogalactopyranoside(IPTG)(aMRESCO,Ohio,USA)使其最終濃度為1mM，置於37℃振盪培養箱誘導4小時至6小時後，轉形株即可表現rSpaA-N重組蛋白。之後，之後，上述誘導所得的rSpaA-N重組蛋白可利用後續SDS-PAGE分析其大小與表現量。 A single colony of the recombinant protein transformed ( E. coli BL 21/rSpaA-N) successfully transformed and cloned was selected and inoculated into 3 mL of LB (Difco, MD, USA; ampicillin with a concentration of 100 μg/mL) The culture solution was shake cultured in a 37 ° C incubator. When the absorbance of the bacterial solution reached OD 600nm 0.6 to 0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) (aMRESCO, Ohio, USA) was added to give a final concentration of 1 mM, and was placed in a shaking incubator at 37 ° C for 4 hours. After 6 hours, the transformed strain can express the rSpaA-N recombinant protein. Thereafter, the rSpaA-N recombinant protein obtained by the above induction can be analyzed for its size and amount by subsequent SDS-PAGE.
4. SDS-PAGE分析4. SDS-PAGE analysis
此實施例使用SE 250 Mini-Vertical Unit for two slab gels電泳槽(GE Healthcare,WI,USA)進行蛋白質分析。首先取兩片製膠玻璃注入已配製完成之8%之下膠體，隨後加入上膠體後，取10μL已製備完成之rSpaA-N重組蛋白至聚丙烯醯胺膠體凹槽內，分別吸取10μL之牛血清蛋白(bovine serum albumin，BSA；KPL,MD,USA)稀釋成500μg/mL、250μg/mL、125μg/mL、62.5μg/mL做為定量標準品，以80伏特進行蛋白質電泳分析。膠體經Coomassie blue染色及分析後，以線性回歸公式算出相對rSpaA-N重組蛋白之濃度，其結果如圖2所示，其中rSpaA-N重組蛋白大小約69KDa，rSpaA-N重組蛋白之濃度則約78μg/mL。 This example used a SE 250 Mini-Vertical Unit for two slab gels electrophoresis tank (GE Healthcare, WI, USA) for protein analysis. First, take two glue injecting glass has completed preparation of colloidal below 8%, followed by the addition of colloidal taken within 10 μ L of completed preparation of the recombinant protein to rSpaA-N polyacrylamide colloid grooves were drawn 10 μ L of bovine serum albumin (bovine serum albumin, BSA; KPL , MD, USA) diluted to 500 μ g / mL, 250 μ g / mL, 125 μ g / mL, 62.5 μ g / mL as a quantitation standard, Protein electrophoresis analysis was performed at 80 volts. After the colloid was stained and analyzed by Coomassie blue, the relative rSpaA-N recombinant protein concentration was calculated by linear regression formula. The results are shown in Figure 2. The rSpaA-N recombinant protein is about 69KDa, and the concentration of rSpaA-N recombinant protein is about 78 μ g / mL.
5. 西方墨點法(Western blot)分析5. Western blot analysis
上述rSpaA-N重組蛋白進行SDS-PAGE後， 以TE 22 Mini Tank Transfer Unit濕式轉漬電泳槽(GE Healthcare,WI,USA)，將rSpaA-N重組蛋白從SDS-PAGE膠體轉漬至PVDF膜。申言之，首先加入轉漬溶液(Transfer buffer)(25mM Tris,192mM glycine,20%methanol)，以300mA將蛋白質轉印至PVDF膜上(polyvinylidene difluoride membrane,Amersham biosciences,Buckinghamshire,UK)上；一級抗體以1,000倍稀釋之耐過豬丹毒桿菌之小鼠血清於4℃作用16-18小時，二級抗體以6,000倍稀釋之goat anti-mice IgG-HRP(KPL,WA,USA)於37℃感作1小時，最後加入1：1比例之ECL plus western blotting detection reagents(GE Healthcare,Newcastle,UK)A及B液混合均勻後，加至PVDF膜上進行呈色，最後以G Box冷光螢光系統影像分析系統(Syngene,Frederick,MD)進行影像分析，結果如圖3所示，於69KDa處有結合條帶產生，證明rSpaA-N重組蛋白具有抗原性。 After the above rSpaA-N recombinant protein is subjected to SDS-PAGE, The rSpaA-N recombinant protein was transferred from the SDS-PAGE colloid to the PVDF membrane using a TE 22 Mini Tank Transfer Unit wet-flow electrophoresis tank (GE Healthcare, WI, USA). In other words, the transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) was first added, and the protein was transferred to a PVDF membrane (polyvinylidene difluoride membrane, Amersham biosciences, Buckinghamshire, UK) at 300 mA; The antibody was treated with a 1,000-fold dilution of the sera of E. rhizogenes-resistant mice at 4 ° C for 16-18 hours, and the secondary antibody was diluted 6,000-fold with goat anti-mice IgG-HRP (KPL, WA, USA) at 37 ° C. For 1 hour, finally add 1:1 ratio of ECL plus western blotting detection reagents (GE Healthcare, Newcastle, UK), A and B liquids are evenly mixed, and then added to the PVDF film for coloring, and finally with G Box cold light fluorescent system. Image analysis system (Syngene, Frederick, MD) performed image analysis. The results are shown in Figure 3. There is a binding band at 69 KDa, which proves that the rSpaA-N recombinant protein is antigenic.
6. 純化rSpaA-N重組蛋白6. Purification of rSpaA-N recombinant protein
將已成功選殖表現之rSpaA-N重組蛋白利用實施例3第1點的方式進行生產。首先，將2L之含有rSpaA-N重組蛋白的轉型株菌液以6,500rpm離心10分鐘(Sorvall RC-6 Plus,Thermo,Massachusetts,USA)，利用20mL lysis buffer(20mM tris base,pH 8.0,8M urea)回溶菌塊，並以超音波震盪器(Misonix Sonicator S-4000,Misonix,New York,USA)進行破菌，最後以全自動純化 機(ProfiniaTM Protein Purification System,Bio-red)通過鎳離子純化管柱(Bio-ScaleTM Mini ProfinityTM IMAC Cartridges,1mL)進行純化，收集純化後的rSpaA-N重組蛋白。 The rSpaA-N recombinant protein which has been successfully colonized was produced by the method of the first point of Example 3. First, 2 L of the transformed strain containing the rSpaA-N recombinant protein was centrifuged at 6,500 rpm for 10 minutes (Sorvall RC-6 Plus, Thermo, Massachusetts, USA) using 20 mL of lysis buffer (20 mM tris base, pH 8.0, 8 M urea). Back to the lysing block and sterilizing with an ultrasonic oscillator (Misonix Sonicator S-4000, Misonix, New York, USA), and finally fully automated The machine (ProfiniaTM Protein Purification System, Bio-red) was purified by a nickel ion purification column (Bio-ScaleTM Mini ProfinityTM IMAC Cartridges, 1 mL), and the purified rSpaA-N recombinant protein was collected.
接下來，利用透析方式去除純化後的rSpaA-N重組蛋白中多餘的尿素。首先將透析膜(Dialysis tubing，TM)浸漬於EDTA緩衝溶液(1%Na2CO3,1mM EDTA)中，以100℃隔水加熱30分鐘。加入純化後之rSpaA-N重組蛋白，平放於0.85%生理食鹽水燒杯內，依序加入濃度5M、4M、3M、2M、1M尿素溶液，於4℃下進行透析，每階段作用時間為1.5小時，最後置於生理食鹽水透析至隔夜，再將透析後之rSpaA-N重組蛋白進行定量。經透析後之rSpaA-N重組蛋白的含量為1015μg/mL。 Next, the excess urea in the purified rSpaA-N recombinant protein was removed by dialysis. First, a dialysis membrane (Dialysis tubing, TM) was immersed in an EDTA buffer solution (1% Na 2 CO 3 , 1 mM EDTA), and heated at 100 ° C for 30 minutes in water. The purified rSpaA-N recombinant protein was added to a 0.85% physiological saline beaker and sequentially added with 5M, 4M, 3M, 2M, 1M urea solution, and dialyzed at 4 ° C. The duration of each phase was 1.5. After an hour, it was dialyzed against physiological saline overnight until the dialysis rSpaA-N recombinant protein was quantified. The content of the rSpaA-N recombinant protein after dialysis was 1015 μg/mL.
1. 製備rSpaA-N疫苗組成物1. Preparation of rSpaA-N vaccine composition
將上述純化完成之rSpaA-N蛋白與W/O/W雙相油質佐劑，以下述配方均勻混合成3組疫苗組成物以及控制組後，利用小鼠進行免疫評估。 The purified rSpaA-N protein and the W/O/W dual phase oily adjuvant were uniformly mixed into the three groups of vaccine compositions and control groups in the following formula, and then subjected to immunological evaluation using mice.
上述3組以及控制組分述如下。第1組：rSpaA-N(400μg/mL)+水包油包水(W/O/W)雙相油質佐劑(例如ISA 201 VG)；第2組：rSpaA-N(200μg/mL)+W/O/W雙相油質佐劑；第3組：rSpaA-N(100μg/mL)+W/O/W雙相油質佐劑；以及第4組(控制組)：未進行任何 免疫。 The above three groups and the control components are as follows. Group 1: rSpaA-N (400 μ g / mL) + water-oil-water (W / O / W) biphasic oily adjuvant (e.g. ISA 201 VG); Group 2: rSpaA-N (200 μ g / mL) + W / O / W biphasic oily adjuvant; group 3: rSpaA-N (100 μ g / mL) + W / O / W biphasic oily adjuvant; and group 4 (control Group): No immunizations were performed.
2. 小鼠接種試驗2. Mouse inoculation test
2.1 小鼠攻毒效力試驗(I)-抗原決定劑量2.1 Mouse challenge efficacy test (I) - antigen-determined dose
選擇4週齡體重15g至18g的ICR小鼠共40隻，隨機分為以下4組、每組10隻進行接種。每隻小鼠以皮下接種0.1mL的rSpaA-N疫苗組成物[每頭小鼠相當於注射10~40微克(μg)之rSpaA-N]，於基礎免疫後2週，分別以1 MLD(5.2 x 102CFU/mL)之血清型第1a型及第2型的豬丹毒桿菌活菌0.1mL，進行皮下攻毒。攻毒後持續觀察2週，並記錄小鼠每組每日之存活率，其結果如表1所示。 A total of 40 ICR mice weighing 4 g to 18 g at 4 weeks of age were randomly divided into the following 4 groups, 10 in each group. Each mouse was subcutaneously inoculated with 0.1 mL of rSpaA-N vaccine composition [equivalent to 10-40 μg (μg) of rSpaA-N per mouse), 2 MLD after basal immunization, respectively, 1 MLD (5.2 x 10 2 CFU/mL) serotypes Type 1a and Type 2 of E. sinensis live bacteria 0.1 mL, for subcutaneous challenge. After the challenge, the observation was continued for 2 weeks, and the daily survival rate of each group of the mice was recorded. The results are shown in Table 1.
由表1之結果顯示，利用第2組配方(rSpaA-N 200μg/mL)及第3組配方(rSpaA-N 100μg/mL)之疫苗組成物進行接種的小鼠，再經血清型第1a型及第2型的豬丹毒桿菌活菌攻毒後，其存活率皆為100%且無任何不良反應，證實具有交叉保護力。因此，以下試驗就以100μg/mL之rSpaA-N重組蛋白作為抗原決定劑量，進行各項免疫效力的評估。 From the results of Table 1 show, using the Group 2 formulation (rSpaA-N 200 μ g / mL) and the third group of formula (rSpaA-N 100 μ g / mL) of the vaccine composition was vaccinated mice, then sera After challenged with type 1a and type 2 of E. sinensis, the survival rate was 100% and there was no adverse reaction, which confirmed cross-protection. Thus, the following experimentation to 100 μ g / rSpaA-N recombinant proteins mL dose was determined as the antigen to evaluate the efficacy of immunization.
2.2小鼠攻毒效力試驗(II)-比較不同血清型之保護效力2.2 Mouse challenge efficacy test (II) - compare the protective efficacy of different serotypes
如上所述，以純化完成之100μg/mL之rSpaA-N重組蛋白作為抗原決定劑量，與W/O/W雙相油質佐劑混合後，利用小鼠比較實施例3之rSpaA-N疫苗組成物與市售疫苗對不同血清型之保護效力。 As described above, in order to complete the purification of the 100 μ g / rSpaA-N recombinant protein as determined mL dose of antigen, the W / O / W After biphasic oily adjuvant, Comparative Example 3 using the mouse rSpaA-N The protective efficacy of vaccine compositions and commercially available vaccines against different serotypes.
選擇4週齡15g至18g的ICR小鼠共30隻，隨機分為3組，每組10隻進行接種。免疫組的每隻小鼠以皮下接種0.1mL的疫苗組成物，控制組的小鼠則未進行任何免疫。於基礎免疫後2週，分別以1 MLD(1.2 x 102CFU/mL)之同源血清型第1a型之豬丹毒桿菌活菌0.1mL，或者以1 MLD(9.2 x 101CFU/mL)之異源血清型第2型之豬丹毒桿菌活菌0.1mL，進行皮下攻毒。攻毒後持續觀察2週，並記錄小鼠每組每日之存活率。 A total of 30 ICR mice of 4 weeks and 15 g to 18 g were selected and randomly divided into 3 groups, and 10 mice in each group were inoculated. Each mouse in the immunized group was subcutaneously inoculated with 0.1 mL of the vaccine composition, and the mice in the control group were not immunized. Two weeks after the basal immunization, 1 MLD (1.2 x 10 2 CFU/mL) homologous serotype 1a type of vivax serotypes 0.1 mL, or 1 MLD (9.2 x 10 1 CFU/mL) The heterologous serotype type 2 of the bacterium was infected with E. sinensis 0.1 mL for subcutaneous challenge. The challenge was continued for 2 weeks after challenge, and the daily survival rate of each group of mice was recorded.
上述6組分述如下。第1、4組：rSpaA-N(100μg/mL)+W/O/W佐劑；第2、5組：市售疫苗〔Swine Erysipelas inactivated bacterin(serotype 1a)，全亞洲製藥廠股份有限公司，台灣〕；第3、6組(控制組)：未進行任何免疫。 The above 6 components are described below. Group 1, 4: rSpaA-N (100 μ g / mL) + W / O / W adjuvant; Group 2, 5: commercial vaccine [Swine Erysipelas inactivated bacterin (serotype 1a) , Asia pharmaceutical limited stock Company, Taiwan]; Groups 3 and 6 (Control Group): No immunizations were performed.
由表2的結果顯示，以同源血清型第1a型的豬丹毒桿菌進行皮下攻毒，第1組(事先以本發明之rSpaA-N 100μg/mL進行免疫)的存活率為100%且無任何不良反應，第2組(事先以市售疫苗進行免疫)僅有60%之存活率，而第3組(控制組)則全數發病死亡(存活率0%)。類似地，以異源血清型第2型的豬丹毒桿菌進行皮下攻毒，第4組(事先以本發明之rSpaA-N 100μg/mL進行免疫)的存活率為100%且無任何不良反應，第5組(事先以市售疫苗進行免疫)及第6組(控制組)則全數發病死亡(存活率0%)。 From the results in Table 2 show, the homologous serotype of type 1a erysipelas subcutaneous challenge, Group 1 (prior to the present invention rSpaA-N 100 μ g / mL immunization) survival rate was 100% There was no adverse reaction, and the second group (previously immunized with a commercially available vaccine) had a survival rate of only 60%, while the third group (control group) had a total morbidity (survival rate of 0%). Similarly, in heterologous serotype 2 of the subcutaneous erysipelas challenge, Group 4 (prior to the present invention rSpaA-N 100 μ g / mL immunization) and the survival rate was 100% without any adverse The reaction, the fifth group (previously immunized with a commercially available vaccine) and the sixth group (control group) all died of morbidity (survival rate 0%).
3. 評估小鼠之血清陽轉率3. Assess the seroconversion rate of mice
選擇4週齡15g至18g的ICR小鼠共30隻，隨機分為3組，每組10隻進行接種。免疫組的每隻小鼠以皮下接種0.1mL的疫苗組成物，於基礎免疫後2週補強注射一劑量，並於免疫前(第0週)、基礎免疫後2週(第2週)、補強接種後2週(第4週)，分別採集小鼠血清。 A total of 30 ICR mice of 4 weeks and 15 g to 18 g were selected and randomly divided into 3 groups, and 10 mice in each group were inoculated. Each mouse in the immunized group was subcutaneously inoculated with 0.1 mL of the vaccine composition, and a dose was given 2 weeks after the basic immunization, and before immunization (week 0), 2 weeks after the basic immunization (week 2), and reinforcement Two weeks after the inoculation (week 4), mouse serum was collected separately.
上述3組分述如下。第1組：rSpaA-N(100μg/mL)+W/O/W佐劑；第2組：市售疫苗〔Swine Erysipelas inactivated bacterin(serotype 1a)，全亞洲製藥廠股份有限公司，台灣〕；第3組(控制組)：未進行任何免疫。 The above three components are as follows. Group 1: rSpaA-N (100 μ g / mL) + W / O / W adjuvant; Group 2: the commercial vaccine [Swine Erysipelas inactivated bacterin (serotype 1a) , Asia Pharmaceutical Co., Ltd., Taiwan] Group 3 (control group): no immunization was performed.
上述所得之小鼠全血以3,000 xg離心10分鐘後，分離血清並保存於-20℃備用。之後，利用下述之酵素連結免疫吸附法(enzyme-linked immunosorbent assay,ELISA)，進一步檢測小鼠血清中IgG、IgG2a與IgG1抗體 力價，其結果分別如圖4至圖6所示。 After the mouse whole blood obtained above was centrifuged at 3,000 x g for 10 minutes, the serum was separated and stored at -20 ° C until use. Thereafter, the following enzyme-linked immunosorbent assay (ELISA) was used to further detect IgG, IgG2a and IgG1 antibodies in mouse serum. The price, the results are shown in Figure 4 to Figure 6, respectively.
3.1 ELISA3.1 ELISA
ELISA係利用以下方式進行。首先，將純化的rSpaA-N重組蛋白塗鍍與coating buffer(15mM Na2CO3,35mM NaHCO3,3mM NaN3,pH 9.6)均勻混合後，平均塗渡於96孔ELISA盤中，置於4℃感作14小時至16小時。一級抗體以1：250倍稀釋之小鼠待測血清，二級抗體分別以1：12000倍稀釋之goat anti-mouse Total IgG-HRP(CALTAG,CA,USA),goat anti-mouse IgG1-HRP(CALTAG,CA,USA),goat anti-mouse IgG2a-HRP(CALTAG,CA,USA)，最後以ELISA reder(Anthos 2020,Cambridge,UK)於OD 450nm測定其吸光值。以S/P ratio計算公式為(實驗組OD值-陰性對照組OD值)/(陽性對照組OD-減陰性對照組OD值)。所得之數據以Tukey significant test分析並以Mean±SEM呈現。圖4至圖6中不同字母代表各組間具有差異性(p<0.05)。 The ELISA was carried out in the following manner. First, the purified rSpaA-N recombinant protein was uniformly mixed with coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , 3 mM NaN 3 , pH 9.6), and then uniformly applied to a 96-well ELISA plate and placed in 4 °C feels 14 hours to 16 hours. The primary antibody was diluted 1:250-fold in the serum to be tested, and the secondary antibody was diluted 1:1000-fold in goat anti-mouse Total IgG-HRP (CALTAG, CA, USA), goat anti-mouse IgG1-HRP ( CALTAG, CA, USA), goat anti-mouse IgG2a-HRP (CALTAG, CA, USA), and finally its absorbance was determined by ELISA reder (Anthos 2020, Cambridge, UK) at OD 450 nm. The formula for calculating the S/P ratio was (OD value of the experimental group - OD value of the negative control group) / (OD value of the negative control group of the positive control group). The data obtained were analyzed by Tukey significant test and presented as Mean ± SEM. The different letters in Figures 4 to 6 represent differences between groups ( p < 0.05).
請參閱圖4，其係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG之抗體力價直條圖。直條401代表第1組(rSpaA-N+W/O/W)之IgG抗體力價，直條403代表第2組(市售疫苗組)之IgG抗體力價，直條405代表第3組(控制組)之IgG抗體力價。 Please refer to FIG. 4, which is a bar graph showing the antibody titer of IgG in the serum after inoculation of the vaccine composition of the sample of the anti-P. sinensis infection of the first embodiment of the invention according to an embodiment of the present invention. The straight bar 401 represents the IgG antibody titer of the first group (rSpaA-N+W/O/W), the straight bar 403 represents the IgG antibody titer of the second group (commercial vaccine group), and the straight bar 405 represents the third group. (Control group) IgG antibody titer.
圖4之IgG抗體力價試驗結果顯示，第1組(rSpaA-N+W/O/W)401於免疫後第2及4週較第2組(市售 疫苗組)403及第3組(控制組)405具顯著性差異(p<0.05)，代表第1組(rSpaA-N+W/O/W)401可以提供較佳的體液性免疫。 The IgG antibody titer test results in Figure 4 show that Group 1 (rSpaA-N+W/O/W) 401 was compared with Group 2 (commercial vaccine group) 403 and Group 3 at 2 and 4 weeks after immunization ( Control group) 405 had a significant difference ( p < 0.05), indicating that Group 1 (rSpaA-N+W/O/W) 401 could provide better humoral immunity.
請參閱圖5，其係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG2a之抗體力價直條圖。直條501代表第1組(rSpaA-N+W/O/W)之IgG2a抗體力價，直條503代表第2組(市售疫苗組)之IgG2a抗體力價，直條505代表第3組(控制組)之IgG2a抗體力價。 Please refer to FIG. 5, which is a bar graph showing the antibody titer of IgG2a in the serum of the mouse after vaccination with the vaccine composition of the anti-P. sinensis infection of the first embodiment according to an embodiment of the present invention. Straight bar 501 represents the IgG2a antibody titer of group 1 (rSpaA-N+W/O/W), bar 503 represents the IgG2a antibody titer of group 2 (commercial vaccine group), and bar 505 represents group 3 (Control group) IgG2a antibody titer.
圖5之IgG2a抗體力價試驗結果顯示，第1組(rSpaA-N+W/O/W)501於免疫後第2及4週較第2組(市售疫苗組503及第3組(控制組)505具顯著性差異(p<0.05)，代表第1組(rSpaA-N+W/O/W)501可以提供較佳的細胞性免疫。 The IgG2a antibody titer test results in Figure 5 show that Group 1 (rSpaA-N+W/O/W) 501 was compared to Group 2 at 2 and 4 weeks after immunization (commercial vaccine group 503 and Group 3 (Control Group 505 had a significant difference ( p < 0.05), indicating that Group 1 (rSpaA-N+W/O/W) 501 could provide better cellular immunity.
請參閱圖6，其係繪示根據本發明一實施例之小鼠利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG1之抗體力價直條圖。直條601代表第1組(rSpaA-N+W/O/W)之IgG1抗體力價，直條603代表第2組(市售疫苗組)之IgG1抗體力價，直條605代表第3組(控制組)之IgG1抗體力價。 Please refer to FIG. 6 , which is a bar graph showing the antibody titer of IgG1 in the serum of the mouse after vaccination with the vaccine composition of the first embodiment of the invention according to an embodiment of the present invention. Straight 601 represents the IgG1 antibody titer of Group 1 (rSpaA-N+W/O/W), straight 603 represents the IgG1 antibody titer of Group 2 (commercial vaccine group), and straight 605 represents Group 3 (Control group) IgG1 antibody titer.
圖6之IgG1抗體力價試驗結果顯示，第1組(rSpaA-N+W/O/W)601於免疫後第2及4週較第2組(市售疫苗組603及第3組(控制組)605具顯著性差異(p<0.05)，代表第1組(rSpaA-N+W/O/W)601可以提供較佳的體液 性免疫。 The IgG1 antibody titer test results in Figure 6 show that Group 1 (rSpaA-N+W/O/W) 601 was compared to Group 2 at 2 and 4 weeks after immunization (commercial vaccine group 603 and Group 3 (control) Group 605 had a significant difference ( p < 0.05), indicating that Group 1 (rSpaA-N+W/O/W) 601 could provide better humoral immunity.
4. 豬隻接種試驗4. Pig inoculation test
4.1安全試驗4.1 Safety test
選擇4週齡抗體陰性健康LYD仔豬8隻，免疫組4隻肌肉注射疫苗2mL[rSpaA-N(100μg/mL)+w/o/w]，並在四週後補強免疫2mL，另4隻則不進行免疫以作為對照組。注射後觀察二週，所有豬隻均無不良反應。 Eight 4-week-old antibody-negative healthy LYD piglets were selected, and 4 mice in the immunized group were injected with 2 mL of the vaccine [rSpaA-N (100 μg/mL) + w/o/w], and after 2 weeks, 2 mL of the booster was immunized, and the other 4 were not. Immunization was performed as a control group. Two weeks after the injection, all pigs had no adverse reactions.
4.2 ELISA4.2 ELISA
所有豬隻於免疫前及免疫後每週採血，分離血清後，以ELISA進行抗體分析。上述所得之豬隻全血以2,500 xg離心10分鐘後，分離血清並保存於-20℃備用。關於ELISA係參照以上第3.1點方式進行，二級抗體是以1：12000倍稀釋之goat anti-swine Total IgG-HRP(CALTAG,CA,USA)，於OD 450nm測定其吸光值，結果如圖7所示。圖7中不同字母代表各組間具有差異性(p<0.05)。 All pigs were bled weekly before immunization and after immunization. After separating the serum, antibody analysis was performed by ELISA. After the whole blood of the pigs obtained above was centrifuged at 2,500 xg for 10 minutes, the serum was separated and stored at -20 ° C until use. The ELISA was performed according to the above point 3.1. The secondary antibody was diluted 1:12000-fold in goat anti-swine Total IgG-HRP (CALTAG, CA, USA), and its absorbance was measured at OD 450 nm. Shown. The different letters in Figure 7 represent differences between groups ( p < 0.05).
請參閱圖7，其係繪示根據本發明一實施例之豬隻利用實施例一之抗豬丹毒桿菌感染之疫苗組成物接種後血清中的IgG之抗體力價直條圖。直條701代表第1組(rSpaA-N+W/O/W)之IgG抗體力價，直條705代表第3組(控制組)之IgG抗體力價。 Please refer to FIG. 7 , which is a bar graph showing the antibody titer of IgG in the serum after inoculation of the vaccine composition of the pig anti-P. sinensis infection of the first embodiment according to an embodiment of the present invention. The straight bar 701 represents the IgG antibody titer of the first group (rSpaA-N+W/O/W), and the straight bar 705 represents the IgG antibody titer of the third group (control group).
圖7之IgG抗體力價試驗結果顯示，第1組(rSpaA-N+W/O/W)701於免疫後第1、2及4週較第3組(控制組)705具顯著性差異(p<0.05)，代表第1組(rSpaA-N+ W/O/W)701可以提供較佳的體液性免疫。 The IgG antibody titer test results in Figure 7 show that Group 1 (rSpaA-N+W/O/W) 701 was significantly different from Group 3 (control group) 705 at 1st, 2nd and 4th week after immunization ( p < 0.05), representing Group 1 (rSpaA-N+ W/O/W) 701 can provide better humoral immunity.
4.3豬隻攻毒效力試驗4.3 Pig challenge test
上述豬隻於補強免疫後2週，分別以1 MLD(6.3 x 108CFU/mL)之同源血清型第1a型之豬丹毒桿菌活菌0.1mL進行皮下攻毒。攻毒後持續觀察2週，並記錄豬隻每組每日之臨床積分。另外，在實驗結束後，所有豬隻犧牲進行剖檢，以記錄豬隻每組之剖檢積分。 The above pigs were subcutaneously challenged with 1 MLD (6.3 x 10 8 CFU/mL) of homologous serotype 1a type of E. sinensis live bacteria 0.1 mL, respectively, 2 weeks after the booster immunization. Continue to observe for 2 weeks after challenge, and record the daily clinical scores of each group of pigs. In addition, at the end of the experiment, all pigs were sacrificed for necropsy to record the necropsy scores for each group of pigs.
免疫組之臨床積分及剖檢積分如表3所示，其中免疫組之臨床症狀及解剖病變積分，較控制組減輕55%及44%，確實具有保護效力。相較之下，控制組的豬隻出現發燒的情況，體溫最高到41.7℃，並出現紅斑症。 The clinical scores and necropsy scores of the immunization group are shown in Table 3. The clinical symptoms and anatomical lesion scores of the immunized group were 55% and 44% less than the control group, and indeed had protective effects. In contrast, pigs in the control group developed fever, with a body temperature of up to 41.7 ° C and erythema.
綜言之，由上述數個實施例證實，本發明以原核表現系統表現rSpaA-N重組蛋白作為動物疫苗組成物，經小鼠試驗證實具有保護受免疫之對象免於至少二不同血清型之豬丹毒桿菌之感染之功效。在其他實施例中，當上述疫苗組成物應用於豬隻時，以三至四週齡的豬隻(體重約4公斤至6公斤)為例，每頭可施打含有200微克(μg)之rSpaA-N重組蛋白的動物用疫苗組成物，其結果與小鼠攻毒試驗相近，亦證實rSpaA-N重組蛋白具有保護受免疫之對象免於至少二不同血清型之豬丹毒桿菌之感染之功效。 In summary, the above embodiments demonstrate that the present invention expresses the rSpaA-N recombinant protein as an animal vaccine composition by a prokaryotic expression system, and has been confirmed by a mouse test to protect a subject who is immunized from at least two different serotypes. The efficacy of the infection with erysipelas. In other embodiments, when the vaccine composition is applied to a pig, a pig of three to four weeks old (body weight of about 4 kg to 6 kg) is used as an example, and each head can be used to contain 200 micrograms (μg) of rSpaA. The animal composition of the -N recombinant protein was similar to the mouse challenge test, and it was also confirmed that the rSpaA-N recombinant protein has an effect of protecting the infected subject from infection by at least two different serotypes of E. sinensis.
需補充的是，本發明雖以特定組成比例的rSpaA-N重組蛋白與W/O/W雙相油質乳劑、特定的製程、特定的分析方法或特定儀器作為例示，說明本發明之交叉保護性重組抗原及含彼之動物疫苗組成物，惟本發明所屬技術領域中任何具有通常知識者可知，本發明並不限於此，在不脫離本發明之精神和範圍內，本發明之交叉保護性重組抗原及含彼之動物疫苗組成物亦可使用其他組成比例的rSpaA-N重組蛋白與W/O/W雙相油質乳劑、其他的製程、其他分析方法或其他儀器進行。 It should be noted that although the present invention exemplifies a specific composition ratio of rSpaA-N recombinant protein and W/O/W dual phase oil emulsion, a specific process, a specific analytical method or a specific instrument, the cross protection of the present invention is illustrated. The present invention is not limited thereto, and the cross-protection of the present invention is not limited thereto, and the present invention is not limited thereto, and the present invention is not limited thereto, and the present invention is not limited thereto, and the cross-protection of the present invention is possible without departing from the spirit and scope of the present invention. Recombinant antigens and animal vaccine compositions containing the same may also be carried out using other constituent ratios of rSpaA-N recombinant protein and W/O/W dual phase oil emulsions, other processes, other analytical methods, or other means.
由上述實施例可知，本發明的交叉保護性重組抗原及含彼之動物疫苗組成物，其優點在於利用豬丹毒桿菌 之表面保護抗原N端片段(rSpaA-N)之重組蛋白作為抗原，以製得動物用次單元疫苗組成物，可誘發至少一種受免疫動物之體內產生抗體，同時刺激細胞性及體液性免疫反應，以保護受免疫動物免於至少二不同血清型之豬丹毒桿菌之感染。 It can be seen from the above examples that the cross-protective recombinant antigen of the present invention and the animal vaccine composition containing the same have the advantages of using the bacterium The recombinant protein of the surface protective antigen N-terminal fragment (rSpaA-N) is used as an antigen to prepare an animal subunit vaccine composition, which can induce the production of antibodies in at least one immunized animal, and stimulate cellular and humoral immune responses. To protect infected animals from at least two different serotypes of E. sinensis infection.
雖然本發明已以數個實施例揭露如上，然其並非用以限定本發明，在本發明所屬技術領域中任何具有通常知識者，在不脫離本發明之精神和範圍內，當可作各種之更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.
<110> 國立屏東科技大學 <110> National Pingtung University of Science and Technology
<120> 交叉保護性重組抗原生物型佐劑及含彼之動物疫苗組成物 <120> Cross-protective recombinant antigen biotype adjuvant and animal vaccine composition containing the same
<130> <130>
<160> 3 <160> 3
<210> 1 <210> 1
<211> 1252 <211> 1252
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 豬丹毒桿菌之表面保護抗原基因第1至1252個核苷酸 <223> The surface protective antigen gene of E. sinensis is 1 to 1252 nucleotides
<400> 1 <400> 1
<210> 2 <210> 2
<211> 26 <211> 26
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 正向引子 <223> Forward introduction
<400> 2 <400> 2
<210> 3 <210> 3
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 3 <400> 3
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| CN113388624A (en) * | 2020-09-21 | 2021-09-14 | 浙江理工大学 | Preparation method of swine erysipelas SpaA antigen protein and optimized clone thereof |
Citations (1)
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| CN101031647A (en) | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
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| CN101031647A (en) | 2004-02-27 | 2007-09-05 | 财团法人化学及血清疗法研究所 | Process for producing erysipelothrix rhusiopathiae surface protective antigen mutant in escherichia coli |
Non-Patent Citations (4)
| Title |
|---|
| DB：ID EM_PRO:KR606300/ KR606288,Source: Erysipelothrix rhusiopathiae strain IMT8949/IMT6245 SpaA gene, complete sequence, 11-DEC-2015 updated into EMBL-Bank. |
| Imada Y et al,"Truncated surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae serotype 1a elicits protection against challenge with serotypes 1a and 2b in pigs.", Infect Immun. 1999 Sep;67(9):4376-82. |
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| CN113388624A (en) * | 2020-09-21 | 2021-09-14 | 浙江理工大学 | Preparation method of swine erysipelas SpaA antigen protein and optimized clone thereof |
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