CN114369152B - 一种重组鸡白细胞介素-9蛋白及其抗体的制备与应用 - Google Patents
一种重组鸡白细胞介素-9蛋白及其抗体的制备与应用 Download PDFInfo
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Abstract
本发明涉及一种重组鸡白细胞介素‑9蛋白及其抗体的制备与应用,本发明要解决的技术问题是制备出具有生物学功能的重组鸡IL‑9原核蛋白与真核蛋白,并测定其生物学活性。本发明还需解决的技术问题是制备检测鸡IL‑9的单克隆和多克隆抗体。并利用这些抗体实现检测鸡体内免疫细胞表达的天然IL‑9蛋白。本发明成功解决了鸡IL‑9细胞因子重组表达难的问题,重组的鸡IL‑9蛋白具有激活鸡巨噬细胞及促进鸡T细胞增殖的生物学活性,提示其具有免疫增强功能。本发明中的鸡IL‑9蛋白及抗体易于大量生产,便于后期在养鸡业和家禽免疫学基础研究中的应用。
Description
技术领域
本发明涉及一种重组鸡白细胞介素-9蛋白及其抗体的制备与应用,具体涉及重组蛋白表达、单克隆及多克隆抗体的制备及其应用,属于禽免疫学、生物制品和诊断试剂领域。
背景技术
白细胞介素-9(IL-9)是一种多效性细胞因子,与IL-2、IL-4、IL-7、IL-15及IL-21同属于γ-链受体细胞因子家族[1]。IL-9最初发现是一种Th2型T细胞分泌的生长因子,但是后来发现主要由Th9型T细胞分泌[2,3];除此之外,Th17、调节性T细胞、CD8+T细胞、自然杀伤细胞、肥大细胞及先天性淋巴样细胞均可分泌IL-9[4]。IL-9可作用于肥大细胞、B细胞、T细胞、造血祖细胞、巨噬细胞、DC细胞等[5]。虽然鸡IL-9基因在鸡的全基因组中有了注释,但在蛋白水平还未被鉴定,其生物学功能仍是空白;其中关键原因是鸡细胞因子的重组表达非常困难以及缺乏特异性检测鸡IL-9蛋白的免疫学工具。
在人和小鼠中,IL-9基因编码一个由144个氨基酸组成的分子量为14kD的糖蛋白,包含一个18个氨基酸的信号肽。然而,天然的IL-9蛋白高度糖基化,分子量约为32到39kD之间[3]。相较之下,鸡IL-9基因编码区包含417个碱基,编码138个氨基酸,包含一个20个氨基酸的信号肽以及3个糖基化位点。但是,由于缺乏相应的检测工具,目前尚不清楚天然的鸡IL-9的真正分子量大小。
IL-9在人或小鼠中具有多种功能。IL-9会诱发自身免疫性疾病,如炎症性肠病、多发性硬化症、类风湿性关节炎等,并在食物过敏和哮喘等过敏性疾病中发挥作用[6]。在癌症方面,IL-9对血液肿瘤和实体瘤有促肿瘤作用,但对黑色素瘤,IL-9和Th9细胞可发挥抗肿瘤活性[7]。此外,IL-9已被证明可以调节免疫应答或在细菌、病毒和寄生虫感染后发挥保护性作用[8]。禽传染性疾病如鸡马立克氏病、传染性法氏囊病、鸡传染性支气管炎等困扰着我国养鸡业的健康发展,鸡IL-9是否具有免疫调节功能、促进禽传染病疫苗的开发以及免疫保护效果有待深入研究。
在本发明中,我们公开了一种具有生物学功能的鸡IL-9真核及原核重组蛋白制备方法;并制备了鸡IL-9鼠源单克隆抗体和兔源多克隆抗体。采用制备的单抗或多抗经Western blot等技术可检测到鸡IL-9天然蛋白。重组鸡IL-9蛋白、单抗及多抗的制备与获得为开发鸡IL-9作为禽传染病的免疫增强剂、佐剂、新型禽病疫苗以及禽免疫学的基础研究提供了重要的工具和手段。
发明内容
本发明的目的是为了解决现有技术的不足,提供重组鸡白细胞介素-9蛋白及其抗体的制备与应用;本发明要解决的技术问题是制备出具有生物学功能的重组chIL-9原核蛋白及真核蛋白,并测定其生物学活性。本发明还需解决的技术问题是制备检测鸡chIL-9的单克隆和多克隆抗体。并利用这些抗体实现检测鸡体内免疫细胞表达的天然chIL-9蛋白。
本发明是通过以下技术方案实现:一种制备重组鸡IL-9蛋白的原核表达方法,不含信号肽的鸡IL-9基因被构建到原核表达载体pET-32a,重组质粒(pET-32a-chIL-9)经转化大肠杆菌诱导表达,获得可溶的重组鸡IL-9蛋白。
一种制备重组鸡IL-9蛋白的原核表达方法,所述不含信号肽的鸡IL-9基因核酸序列如SEQ ID NO.1所示,其氨基酸序列如SEQ ID NO.2所示;
所述重组鸡IL-9蛋白具有生物学活性,将重组鸡IL-9蛋白与鸡巨噬细胞系共培养可激活巨噬细胞产生炎性细胞因子;将重组鸡IL-9蛋白与分离的鸡脾脏细胞共培养5天,可促进鸡T细胞的增殖。
所述的重组鸡IL-9蛋白的应用,可作为免疫增强剂提高禽类,特别是鸡的免疫能力;可作为佐剂用于家禽,特别是鸡传染病疫苗的开发与应用。
一种制备重组鸡IL-9蛋白的真核表达方法,完整的鸡IL-9基因被构建到真核表达载体pEGFP-C,重组质粒(pEGFP-chIL-9)转染DF-1细胞,获得分泌表达的重组鸡IL-9蛋白,并具有生物学活性;
所述的完整鸡IL-9基因核酸序列如SEQ ID NO.3所示,其氨基酸序列如SEQ IDNO.4所示;
将重组鸡IL-9蛋白与鸡巨噬细胞系共培养可激活巨噬细胞产生炎性细胞因子;将重组鸡IL-9蛋白与分离的鸡脾脏细胞共培养5天,可促进鸡T细胞的增殖。
所述的重组鸡IL-9蛋白的应用,可作为免疫增强剂提高禽类,特别是鸡的免疫能力;可作为佐剂用于家禽,特别是鸡传染病疫苗的开发与应用。
一株抗鸡IL-9蛋白单克隆抗体4H7,所述单克隆抗体4H7能够特异性结合鸡免疫细胞表达的IL-9蛋白;能够特异性识别经佛波酯和离子霉素活化的鸡单个核细胞表达的IL-9天然蛋白。
将所述重组鸡IL-9蛋白免疫BALB/c小鼠,通过杂交瘤技术,经所述的真核表达鸡IL-9蛋白筛选而获得的单克隆抗体细胞株,即杂交瘤细胞株4H7;
所述杂交瘤细胞株4H7保藏于中国典型培养物保藏中心,地址为:中国武汉,武汉大学;保藏日期为2022年1月19日;保藏编号为CCTCC NO:C202225。
一种抗鸡IL-9蛋白兔源多克隆抗体的制备方法,将所述重组鸡IL-9蛋白免疫新西兰大白兔,获得的抗血清能够识别真核表达的鸡IL-9蛋白及激活的鸡单个核细胞表达的IL-9天然蛋白。
抗鸡IL-9蛋白单克隆抗体4H7的应用,经荧光素或酶标记后用于Western-blot、ELISA、间接免疫荧光和流式细胞术检测鸡IL-9的表达。
多克隆抗体的应用,经荧光素或酶标记后用于Western-blot、ELISA、间接免疫荧光和流式细胞术检测鸡IL-9的表达。
本发明方法先进、科学,通过本发明,提供了一种重组鸡白细胞介素-9蛋白及其抗体的制备与应用,构建pET-32a-chIL-9和pEGFP-C-chIL-9质粒,并分别在ROSETTA菌和DF-1细胞中表达chIL-9重组蛋白;用qPCR法和CFSE染色法分别检测重组chIL-9对鸡巨噬细胞激活及T细胞增殖的作用。重组chIL-9原核蛋白免疫BALB/c小鼠三次,取脾脏与SP2/0细胞融合并筛选出阳性杂交瘤细胞,对不同杂交瘤细胞株分泌的抗体进行chIL-9真核蛋白鉴定,并制备腹水、纯化抗体、western blot技术测定不同抗体与chIL-9天然蛋白的反应性。
有益效果:
本发明成功解决了鸡IL-9细胞因子重组表达难的问题,重组的chIL-9蛋白具有激活鸡巨噬细胞及促进鸡T细胞增殖的生物学活性,提示其具有免疫增强功能。本发明成功制备了抗chIL-9单克隆和多克隆抗体,并能高效特异性识别chIL-9天然蛋白。本发明填补了鸡IL-9蛋白的生物学功能和免疫检测知识和技术空白,并为chIL-9在鸡免疫抑制性疾病中的应用提供了有效工具与生物制剂。本发明中的chIL-9蛋白及抗体易于大量生产,便于后期在养鸡业和家禽免疫学基础研究中的应用。
附图说明
图1为pET-32a-chIL-9质粒的构建,包括chIL-9PCR扩增(a)、质粒图谱(b)及质粒酶切鉴定(c);M:DNA Marker;1:chIL-9;2:pET-32a-chIL-9质粒双酶切产物。
图2为chIL-9原核表达(a),蛋白纯化(b),复性、标签蛋白切除(c);a图,M:Prestained Protein Ladder;1:control组裂解上清2:IPTG组裂解上清3.control组裂解沉淀4.IPTG组裂解沉淀。b图,M:Prestained Protein Ladder;5:IPTG组裂解沉淀6:纯化后的Trx-chIL-9蛋白。c图,M:Prestained Protein Ladder;7:tev酶切割后的chIL-9。
图3为pEGFP-C-chIL-9质粒的构建,包括chIL-9PCR扩增(a)、质粒图谱(b)及质粒酶切鉴定(c);M:DNA Marker 1:chIL-9-CDS;2:pEGFP-C-chIL-9质粒双酶切产物。
图4为chIL-9真核表达的鉴定,包括ICC(a)和western blot(b);a图,A:pEGFP-C+阴性血清B:pEGFP-C+chIL-9多抗血清C:pEGFP-C-chIL-9+阴性血清D:pEGFP-C-chIL-9+chIL-9多抗血清。b图,M:Prestained Protein Ladder 1:pEGFP-C转染的DF-1 2:pEGFP-C-chIL-9转染的DF-1 3:pEGFP-C转染的DF-1培养上清4:pEGFP-C-chIL-9转染的DF-1培养上清。
图5为重组chIL-9真核蛋白对单核巨噬细胞炎症因子产生的影响;a图,chIL-9对HD11炎症因子产生的影响;b图,chIL-9对原代单核细胞炎症因子长生的影响。
图6为重组chIL-9蛋白对CD3+ T细胞增殖的影响,包括原核和真核重组chIL-9蛋白。
图7为4株chIL-9单抗对真核表达chIL-9的识别。
图8为chIL-9鼠单抗和兔多抗与天然chIL-9蛋白反应图。
具体实施方式
为了全面了解和应用本发明,下文将给出参考实施例和附图详细讲述本发明。
实施例1chIL-9重组蛋白制备
1.chIL-9原核蛋白表达
由于鸡的基因密码子与大肠杆菌宿主适应性低,首先把克隆的不含信号肽的chIL-9核苷酸序列(如SEQ ID NO.1所示),优化成大肠杆菌使用频率较高的密码子。使用Primer5.0软件设计PCR引物,并引入酶切位点BamH Ⅰ和Xho Ⅰ和tev蛋白酶切位点,进行RT-PCR扩增,并克隆到原核表达载体pET-32a中(图1),最终转化至ROSETTA表达宿主菌中。
PCR引物序列(SEQ ID NO.5和6所示):
F:5’-CGGGATCC(BamHI)GAGAACCTGTACTTCCAAGGG(tev)CAGAATTGCCAGGTT-3’
R:5’-CCGCTCGAG(XhoI)TCACACGCGGCTTTTAT-3’.
含目的基因的重组质粒转化ROSETTA宿主菌后,经IPTG小量诱导,分别提取细菌裂解上清蛋白和菌体蛋白;经SDS-PAGE分析发现chIL-9重组融合蛋白大量表达于细菌裂解沉淀中。取300mL菌液经IPTG大量诱导后,冰浴超声20~30min,离心去上清,10mL 8M尿素重悬和分散沉淀,冰浴下再超声10min,离心后收集上清。根据Ni Sepharose柱说明书进行变性条件下的his标签蛋白的纯化,纯化后的蛋白经过梯度尿素缓冲液进行透析复性,最后进行浓缩以及蛋白浓度测定。用tev酶切掉trx标签可得到约为18kD的chIL-9原核重组蛋白(图2)。
2.chIL-9真核蛋白表达
取鸡脾单个核细胞,经PMA/Iono+BFA刺激6h后,提取RNA,并反转录成cDNA作为模板。根据chIL-9基因全长编码区序列(如SEQ ID NO.3所示)设计引物,在上游引物前端加入T2A肽序列,并引入EcoRI和BamHI酶切位点,进行RT-PCR扩增,并克隆到pEGFP-C质粒(图3),最终提取出超纯质粒。
PCR引物序列(SEQ ID NO.7和8所示):
F:5’-CCGGAATTC(EcoRI)GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCTATGAATGCCAGCATGCTG-3’
R:5’-CGGGATCC(BamHI)TTAAACTCTAGATTTATG-3’
将目的质粒转染至DF-1细胞内,经细胞免疫化学(ICC)和Western blot检测,DF-1细胞内和培养上清中均有25kD的重组chIL-9蛋白产生(图4)。
实施例2chIL-9重组蛋白的生物学功能
1.chIL-9重组真核蛋白对鸡巨噬细胞的激活作用
本试验选取鸡HD11巨噬细胞系及鸡外周血单个核细胞贴壁后的原代巨噬细胞检测chIL-9重组蛋白的功能。对照组添加pEGFP-C质粒转染上清,chIL-9组添加pEGFP-C-chIL-9质粒转染上清,LPS组作为阳性对照;作用6h后提取RNA,反转录成cDNA,qPCR检测巨噬细胞关键活化因子IL-1β、IL-6及iNOS的变化。图5结果表明,重组真核chIL-9可以激活鸡巨噬细胞产生炎性因子。
2.chIL-9重组蛋白对鸡T细胞的增殖作用
本试验选取鸡外周血单个核细胞(PBMC)作为靶细胞检测chIL-9重组蛋白对T细胞的增殖作用。将PBMC染完CFSE后,添加chIL-9重组蛋白(100ng/mL)、鸡IL-2(100ng/mL)作用五天后,做SPRD-CD3及FVD eFluor 780染色。流式细胞术结果表明(图6),重组chIL-9可以促进鸡T细胞增殖。
实施例3chIL-9鼠单克隆抗体的制备
单抗制备的主要步骤有:小鼠免疫、细胞融合、杂交瘤细胞的筛选、细胞单克隆化、抗体的鉴定、腹水制备及纯化。
1.阳性杂交瘤细胞的筛选
选用6周龄雌性BALB/c小鼠,将40μg原核表达的chIL-9与弗氏佐剂混合并乳化。通过腹腔注射对小鼠免疫三次,每次免疫间隔两周,7天后尾静脉采血测定效价后加强免疫,三天后取小鼠脾细胞在50%PEG-1500的作用下与SP2/0细胞融合,并加入到含HAT培养基的饲养层细胞中,放置于37℃,5%CO2细胞培养箱中。5天后半换液,10天后换成含HT的DMEM完全培养液。
用ELISA法测定5块96孔板中的阳性孔,并采取有限稀释法把阳性杂交瘤孔2~3次亚克隆,最后扩大培养,并冻存。收集单抗杂交瘤细胞培养上清,根据小鼠抗体亚型鉴定试剂盒进行亚类鉴定。
2.单抗腹水制备及纯化方法
选取10周龄左右BALB/c小鼠,腹腔接种0.5mL降植烷,7~10天后腹腔接种PBS稀释的杂交瘤细胞,细胞数为5×105/只。7天后腹部膨胀,腹腔采集腹水,每两天一次,将腹水离心去除沉淀物,收集上清,测效价,-70℃保存。使用Protein A+G将腹水纯化。
实施例4chIL-9兔多克隆抗体的制备及纯化
将100μg原核表达的chIL-9与完全弗氏佐剂混合并乳化,皮下多点注射免疫新西兰大白兔;并用重组原核chIL-9与不完全弗氏佐剂的乳化物加强免疫两次,每次间隔两周;7天后静脉采血测定效价后加强免疫(200μg),三天后静脉采血获取抗血清,并用Protein A+G将兔血清纯化,最终获得chIL-9兔多克隆抗体。
实施例5chIL-9单克隆抗体及兔多克隆抗体与真核表达chIL-9反应性
将pEGFP-C-chIL-9质粒转染DF-1细胞内,48h后弃掉培养液,PBS洗两遍,用冰冷的丙酮乙醇混合液(3:2)固定,经2%BSA室温封闭1h后,孵育不同杂交瘤细胞培养上清、IgG1,κ亚型无关鼠单抗(control)及兔多克隆抗体(pAb),4℃过夜孵育,PBST洗3遍,室温孵育HRP偶联的anti-mouse或anti-rabbit抗体1h,PBST洗3遍,加入AEC显色液反应15min,明场条件下观察着色情况。结果(图7,table 1)表明,不同chIL-9单克隆抗体及兔多克隆抗体均与真核chIL-9反应,单抗4H7和其它3株单抗均为IgG1,κ亚型。
实施例6chIL-9单克隆抗体及兔多克隆抗体与天然chIL-9的反应性
取鸡PBMC,经PMA/Iono+BFA刺激6h后,提取蛋白,加入5×loading buffer,煮沸10min后即chIL-9天然蛋白样品。取各组蛋白进行SDS-PAGE电泳,转印至PVDF膜上,5%脱脂乳室温封闭1h,4℃过夜孵育不同单克隆抗体和兔多克隆抗体(1:1000),TBST溶液清洗3遍后,孵育HRP goat anti-mouse二抗(1:8000)37℃1h,TBST洗3遍后加入发光液进行显影。图8结果显示,不同单克隆抗体和兔多克隆抗体与chIL-9天然蛋白反应,大小约为25kDa。
表1 chIL-9鼠单抗亚型鉴定与腹水效价测定
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序列表
<110> 扬州大学
<120> 一种重组鸡白细胞介素-9 蛋白及其抗体的制备与应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 357
<212> DNA
<213> 鸡(Gallus gallus)
<400> 1
cagaattgcc aagtcagcga aattatccgc aatacaagga gtctgcttaa caagactcaa 60
gaaagttgcc catgtaggga gacagctgcc actccttgtt catgtcttcc catccctgag 120
cctggccatg aactggcatg ctttgtggaa ggaaccaagc acatgttgga gcacaacatg 180
acttcaaaca taaaacttct tctgcttaat cagtccttcc agattcaacg ggatagaaac 240
ctctgtgaaa gtctgacacg tggaaaaaaa tgcgagtaca agaccaaggg aactgtgagg 300
acgtttctgg aaaaaatcct gaaaacctat caggaaatcc ataaatctag agtttaa 357
<210> 2
<211> 118
<212> PRT
<213> 鸡(Gallus gallus)
<400> 2
Gln Asn Cys Gln Val Ser Glu Ile Ile Arg Asn Thr Arg Ser Leu Leu
1 5 10 15
Asn Lys Thr Gln Glu Ser Cys Pro Cys Arg Glu Thr Ala Ala Thr Pro
20 25 30
Cys Ser Cys Leu Pro Ile Pro Glu Pro Gly His Glu Leu Ala Cys Phe
35 40 45
Val Glu Gly Thr Lys His Met Leu Glu His Asn Met Thr Ser Asn Ile
50 55 60
Lys Leu Leu Leu Leu Asn Gln Ser Phe Gln Ile Gln Arg Asp Arg Asn
65 70 75 80
Leu Cys Glu Ser Leu Thr Arg Gly Lys Lys Cys Glu Tyr Lys Thr Lys
85 90 95
Gly Thr Val Arg Thr Phe Leu Glu Lys Ile Leu Lys Thr Tyr Gln Glu
100 105 110
Ile His Lys Ser Arg Val
115
<210> 3
<211> 417
<212> DNA
<213> 鸡(Gallus gallus)
<400> 3
atgaatgcca gcatgctgtc atacattctc ctctcttgtg tgctgctctc tgtgcaagca 60
cagaattgcc aagtcagcga aattatccgc aatacaagga gtctgcttaa caagactcaa 120
gaaagttgcc catgtaggga gacagctgcc actccttgtt catgtcttcc catccctgag 180
cctggccatg aactggcatg ctttgtggaa ggaaccaagc acatgttgga gcacaacatg 240
acttcaaaca taaaacttct tctgcttaat cagtccttcc agattcaacg ggatagaaac 300
ctctgtgaaa gtctgacacg tggaaaaaaa tgcgagtaca agaccaaggg aactgtgagg 360
acgtttctgg aaaaaatcct gaaaacctat caggaaatcc ataaatctag agtttaa 417
<210> 4
<211> 138
<212> PRT
<213> 鸡(gallus gallus)
<400> 4
Met Asn Ala Ser Met Leu Ser Tyr Ile Leu Leu Ser Cys Val Leu Leu
1 5 10 15
Ser Val Gln Ala Gln Asn Cys Gln Val Ser Glu Ile Ile Arg Asn Thr
20 25 30
Arg Ser Leu Leu Asn Lys Thr Gln Glu Ser Cys Pro Cys Arg Glu Thr
35 40 45
Ala Ala Thr Pro Cys Ser Cys Leu Pro Ile Pro Glu Pro Gly His Glu
50 55 60
Leu Ala Cys Phe Val Glu Gly Thr Lys His Met Leu Glu His Asn Met
65 70 75 80
Thr Ser Asn Ile Lys Leu Leu Leu Leu Asn Gln Ser Phe Gln Ile Gln
85 90 95
Arg Asp Arg Asn Leu Cys Glu Ser Leu Thr Arg Gly Lys Lys Cys Glu
100 105 110
Tyr Lys Thr Lys Gly Thr Val Arg Thr Phe Leu Glu Lys Ile Leu Lys
115 120 125
Thr Tyr Gln Glu Ile His Lys Ser Arg Val
130 135
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cgggatccga gaacctgtac ttccaagggc agaattgcca ggtt 44
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ccgctcgagt cacacgcggc ttttat 26
<210> 7
<211> 90
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccggaattcg gaagcggaga gggcagagga agtctgctaa catgcggtga cgtcgaggag 60
aatcctggac ctatgaatgc cagcatgctg 90
<210> 8
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgggatcctt aaactctaga tttatg 26
Claims (2)
1.一株抗鸡IL-9蛋白单克隆抗体4H7,其特征在于,所述单克隆抗体4H7能够特异性结合鸡免疫细胞表达的IL-9蛋白;能够特异性识别经佛波酯和离子霉素活化的鸡单个核细胞表达的IL-9天然蛋白;
所述杂交瘤细胞株4H7保藏于中国典型培养物保藏中心,地址为:中国武汉,武汉大学;保藏日期为2022年1月19日;保藏编号为CCTCC NO: C202225。
2.权利要求1所述的抗鸡IL-9蛋白单克隆抗体4H7非诊断与治疗目的的应用,其特征在于,经荧光素或酶标记后用于Western-blot、ELISA、间接免疫荧光和流式细胞术检测鸡IL-9的表达。
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