CN115317599A - 一种绵羊肺炎支原体亚单位疫苗的制备方法 - Google Patents
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Abstract
本发明公开了一种绵羊肺炎支原体亚单位疫苗的制备方法。通过将绵羊肺炎支原体模式菌株Y98的外膜HSP70蛋白C末端编码基因以及绵羊肺炎支原体优化后的多表位抗原meAg5编码基因分别克隆至pET32a(+)构建pET32a(+)‑HSP70和pET32a(+)‑meAg5重组质粒。转入DE3感受态细胞中诱导HSP70和meAg5重组蛋白成功表达并与佐剂乳化制备亚单位疫苗，获得的亚单位疫苗通过动物免疫实验后，可以刺激机体产生较高的抗原特异性免疫反应。与传统灭活疫苗相比本发明生产过程便捷并且成本低廉，减少了支原体的复杂培养过程。
Description
技术领域
本发明涉及一种绵羊肺炎支原体亚单位疫苗的制备方法。
背景技术
羊支原体肺炎(Mycoplasmal pneuoniae ofsheep and goats，MPSG)是由支原体引起的、一种高度接触性传染病，具有高发病率和死亡率，绵羊肺炎支原体是其重要病原之一(Li M,Ma C J,Liu X M,et al.Molecular cloning of HSP70 in Mycoplasmaovipneumoniae and comparison with that ofother mycoplasmas[J].Genet Mol Res,2011,10(2)：834-848.)。绵羊肺炎支原体可感染绵羊、山羊、大角羊和野生小反刍动物，引起致命性肺炎。患病羊只以呼吸系统疾病为主，产生咳嗽、喘气、渐进性消瘦及慢性增生性间质性肺炎等症状(Cheng C,Jun Q,Qingling M,et al.Serological and molecularsurvey of sheep infected with Mycoplasma ovipneumoniae in Xinjiang,China[J].Tropical animal health andproduction,2015,47(8)：1641-1647.)，并且该病还能引起支气管上皮细胞增生以及淋巴细胞或中性粒细胞的额外浸润等相关器官的病变，给国家甚至世界羊养殖业带来了巨大的经济损失(Besser TE,Levy J,Ackerman M,et al.A pilotstudy of the effects of Mycoplasma ovipneumoniae exposure on domestic lambgrowth andperformance[J].PLoS One,2019,14(2)：e0207420.)。
绵羊肺炎支原体(Mycoplasma ovipneumoniae，M.ovipneumoniae，Mo)是支原体家族内支原体科的一种介于细菌与病毒之间无细胞壁结构的原核微生物，具有由环状双链DNA组成的微小基因组，在电镜下可观察到三层膜的细胞膜，从外到内分别为蛋白质、脂质、蛋白质，是能够特异性感染绵羊的支原体细菌(Chen J,Zhou Y,Zhu E,et al.Mycoplasmaovipneumoniae induces caspase-8-dependent extrinsic apoptosis and p53-andROS-dependent intrinsic apoptosis in murine alveolar macrophages[J].Virulence,2021,12(1)：2703-2720.)，绵羊肺炎支原体可侵入呼吸道并寄居在气管上皮细胞，然后产生毒性代谢物过氧化氢，破坏纤毛清除功能，导致严重的混合感染(Zhao J,DuY,Song Y,et al.Investigation ofthe prevalence of in Southern Xinjiang,China[J].Journal of Veterinary Research,2021,65(2)：155-160.)。近年来，随着中国养殖业的发展，由绵羊肺炎支原体引起的绵羊支原体肺炎在西部地区如甘肃、宁夏、新疆等地广泛流行(张轩.绵羊肺炎支原体多表位疫苗的研究及免疫试验[D].中国农业科学院,2013.)。该病不仅一年四季均有发生(秋冬季节发病率较高)，而且不同饲养阶段的羊只均可感染，其中羔羊最为易感、流行，发病率可达90％以上，死亡率接近30％，并且该病危害日趋严重，目前成为危害绵羊的主要传染病之一，给养羊业造成了巨大的经济损失，因此开展绵羊肺炎支原体的疫苗研究刻不容缓。(Goltz J P，Rosendal S，Mccraw B M，etal.Experimental studied on the pathogenicity of Mycoplasma ovipneumoniae andMycoplasma arginini for the respiratory tract of goals[J].Can J Vet Res,1986,1：59-67)目前使用的疫苗多为灭活疫苗，但由于支原体无细胞壁营养需求高、培养条件苛刻、生长缓慢、培养周期长和免疫原性较弱等因素，限制了绵羊肺炎支原体灭活疫苗的研究(Hernandez F J，Stockdale K R，Huang L，et al.Degradation of nuclease-stabilizesd RNA oligonucleotides in Mycoplasma-contaminated cell culturemedia[J].Nucleic Acid Ther，2012，22(1)：58-68.)，因此目前的研究多为建立一种基因工程疫苗来应对羊支原体肺炎。热休克蛋白(HSP70)是包括支原体在内的各种细菌物种中最保守的蛋白之一，是绵羊肺炎支原体的膜相关蛋白对于肺炎支原体与宿主细胞的粘附至关重要(Jiang F,He J,Navarro-Alvarez N,et al.Elongation factor Tu and heatshockprotein70are membrane-associated proteins from Mycoplasma ovipneumoniaecapable of inducing strong immune response in mice[J].PloS one,2016,11(8)：e0161170.)，Li等人研究表明Mo的HSP70的C端可以诱导小鼠产生强烈的血清反应，且可以在免疫应答中充当Th1细胞因子样佐剂，这表明HSP70蛋白可能是针对绵羊肺炎支原体感染的疫苗开发的相关抗原(Li M,Ma C J,Liu X M,et al.Molecular cloning ofHSP70 inMycoplasma ovipneumoniae and comparison with that ofother mycoplasmas[J].Genet Mol Res,2011,10(2)：834-848.)。抗原表位融合技术是研制基因工程亚单位疫苗的重要策略之一，乔军等人研究表明Mo多表位融合抗原Mo-meAg5优化后对小鼠具有较好的免疫原性，可能是Mo疫苗的潜在候选者(乔军，陈诚，孟庆玲等.绵羊肺炎支原体多表位融合抗原MO-meAg5及其制备方法和应用[P].新疆维吾尔自治区：CN105859845B，2019-03-12.)。
因此本发明提出了一种绵羊肺炎支原体亚单位疫苗的制备方法，并通过动物实验检测其免疫原性，为绵羊肺炎支原体基因工程疫苗的开发提供新的解决思路。
发明内容
本发明公开了一种绵羊肺炎支原体亚单位疫苗的制备方法。通过原核表达的方式诱导重组蛋白HSP70和meAg5表达，构建一种绵羊肺炎支原体亚单位疫苗并通过动物实验分析其免疫原性，本发明提供的方法能够便捷快速的获得抗原蛋白，并且能够诱导动物产生较高的免疫反应，预计可以为治疗羊支原体肺炎提供简单有效的方法，为绵羊肺炎支原体疫苗的研究奠定基础。
为了达到上述技术效果，本发明采取以下方案：
本发明利用通过基因体外合成的方式分别合成HSP70和meAg5序列，利用Xho I和EcoR I酶切链接的方法构建pET32a(+)-HSP70和pET32a(+)-meAg5重组质粒，并转化入DE3感受态细胞中进行重组HSP70和meAg5蛋白的诱导表达。
本发明利用镍柱对收集的蛋白液进行纯化并通过超滤管对蛋白进行浓缩，纯化效果利用SDS-PAGE验证并通过BCA蛋白检测试剂盒测定收集的蛋白浓度。
本发明利用表达纯化的重组HSP70和meAg5蛋白两种蛋白制备绵羊肺炎支原体亚单位疫苗。主要实施方法为将纯化后的两种蛋白混合，每份疫苗中含有25ug重组HSP70蛋白和25ug重组meAg5蛋白并与50ug弗氏佐剂混合，使用乳化仪制成疫苗。
本发明制备的亚单位疫苗通过动物实验检测其免疫原性，亚单位疫苗利用皮下注射的方法每只小鼠注射100ul后收集动物血清，再利用ELISA方法检测动物血清中特异性IgG抗体的产生情况，ELISA验证结果表明本发明制备的亚单位疫苗与对照组相比具有良好的免疫原性并产生较高的特异性IgG反应。
本发明具有免疫原性亚单位疫苗的制备方法在绵羊肺炎支原体感染导致的羊支原体肺炎中具有较高的应用价值，绕过了支原体难以培养的步骤并预计可以抵抗不止一种绵羊肺炎支原体的入侵，以期为绵羊肺炎支原体疫苗的研制提供参考。
附图说明
图1是重组pET32a(+)-HSP70和pET32a(+)-meAg5酶切鉴定图；
其中：M：DNAMarker；1：pET32a(+)-HSP70重组质粒；2：pET32a(+)-HSP70重组质粒；3：pET32a(+)-meAg5重组质粒；4：pET32a(+)-meAg5重组质粒。
图2是重组HSP70和meAg5蛋白的SDS-PAGE分析图；
其中：M：蛋白分子质量标准；H1：HSP70重组菌对照组；H2：HSP70重组菌诱导组；H3：HSP70重组菌诱导沉淀组；H4：HSP70重组菌诱导上清组；H5：HSP70重组菌诱导纯化组；E1：me-Ag5重组菌对照组；E2：me-Ag5重组菌诱导组；E3：me-Ag5重组菌诱导沉淀组；E4：me-Ag5重组菌诱导上清组；E5：me-Ag5重组菌诱导纯化组。
图3是免疫小鼠血清中IgG水平的分析结果图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细叙述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
步骤1：重组pET32a(+)-HSP70和pET32a(+)-meAg5原核表达载体的构建
1、目的基因的酶切与回收
通过基因体外合成的方式分别合成带有酶切位点的绵羊肺炎支原体模式菌株Y98的HSP70蛋白C末端序列以及绵羊肺炎支原体优化后的多表位抗原meAg5序列，并将带有目的基因的菌液扩大培养，提取质粒并检测浓度，在37℃的条件下使用设计的酶切位点XhoI和EcoR I对合成的两条基因(目的片段)双酶切60-90分钟后使用1％琼脂糖凝胶电泳验证后用琼脂糖凝胶回收试剂盒回收酶切片段，测浓度，并置于-20℃保存。
2、目的基因和载体的连接、转化
将含有pET32a(+)质粒的菌液扩大培养提取质粒后同样使用XhoI和EcoR I进行双酶切，通过1％琼脂糖凝胶电泳验证后用琼脂糖凝胶回收试剂盒回收酶切片段，将酶切后的两条目的基因与酶切后的pET32a(+)载体进行连接，构建pET32a(+)-HSP70和pET32a(+)-meAg5重组质粒。将连接后的重组质粒转化进入DH5a感受态细胞中筛选阳性克隆，并且提取质粒进行双酶切及测序验证，双酶切验证结果正确，在700bp和400bp左右的位置显示出明显的条带。挑选验证正确的阳性克隆扩大培养并提取质粒，将提取的质粒转入DE3感受态细胞中并进行抗生素筛选，将验证正确的阳性克隆加入50％的甘油并置于-80℃冰箱保存。
步骤2：重组HSP70和meAg5蛋白的表达纯化
1、重组HSP70和meAg5蛋白的诱导表达
将含有pET32a(+)-HSP70和pET32a(+)-meAg5重组质粒的DE3菌株使用千分之二浓度的IPTG诱导6h后，12000rpm/min离心5min收集菌株，加入裂解液后使用涡旋震荡仪混合均匀，4℃静置过夜。将裂解后的菌液在液氮速冻5min然后放置在37℃水浴锅中完全融化，反复冻融3次后利用超声破碎仪进行超声破碎：工作3s，间隔2s，20min/周期，裂解菌体放在冰水混合物中重复3个周期。将超声后的菌液在12000rpm，4℃条件下离心30min，分离上清和沉淀。上清置于新的离心管中，沉淀用8M尿素复溶。分别取上清和沉淀进行SDS-PAGE电泳，鉴定到IPTG可诱导目的蛋白在可溶性的上清中表达。
2、蛋白的纯化和浓缩
将验证过的上清用0.45μm的滤膜进行过滤，将滤液通过镍柱纯化。先用10倍的A液平衡镍柱，然后将蛋白上柱，再用5～10倍的A液平衡镍柱后用10倍的B液洗脱，接取洗脱液，收得蛋白液。然后用10倍的A液平衡柱子，再用5倍柱体积的去离子水洗脱，最后用5倍柱体积的20％的乙醇洗脱。将收集的蛋白液通过SDS-PAGE电泳检测蛋白纯度后将蛋白液放入超滤管中浓缩，用BCA试剂盒测定浓度后分装保存于-80℃冰箱。
步骤3：绵羊肺炎支原体亚单位疫苗的制备
将纯化后的重组HSP70和meAg5蛋白液混合，每只小鼠的量为25ug重组HSP70蛋白和25ug重组meAg5蛋白，并与弗氏佐剂一比一混合，初次免疫的疫苗为蛋白液与弗氏完全佐剂混合乳化，对照组注射PBS，初免后14天再次免疫，此时免疫的疫苗为蛋白液与弗氏不完全佐剂混合乳化，对照组此时也注射相同量的PBS。
步骤4：绵羊肺炎支原体亚单位疫苗的免疫原性研究
ELISA检测免疫小鼠血清中特异性IgG抗体应答
以原核表达纯化的重组HSP70和meAg5蛋白为抗原，每孔包被4ug重组HSP70和4ug重组meAg5蛋白，4℃过夜包被；5％的脱脂奶粉37℃封闭2小时；将0，14，28，42天收集的小鼠血清稀释，加入96孔板中，37℃孵育lh；以HRP标记的羊抗鼠IgG为二抗37℃孵育lh；显色之后使用酶标仪450nm进行检测，ELISA检测结果如图3所示，在免疫前实验组(疫苗免疫)与对照组(PBS)特异性IgG抗体的产生无显著差异(p>0.05)，而从初免后14d开始实验组达到了保护水平(P/N＝4.49)且特异性IgG抗体显著高于对照组(p<0.01)，在二免后14d特异性IgG抗体不断上升到达峰值，虽然在第42d特异性IgG抗体有所下降但始终显著高于对照组(p<0.01)。
Claims (2)
1.一种绵羊肺炎支原体亚单位疫苗的制备方法，其特征在于包括以下步骤：将HSP70和meAg5序列，分别利用XhoI和EcoR I酶切链接的方法构建pET32a(+)-HSP70和pET32a(+)-meAg5重组质粒，并转化入DE3感受态细胞中进行重组HSP70和meAg5蛋白的诱导表达后收集蛋白液；
利用镍柱对收集的蛋白液进行纯化并通过超滤管对蛋白进行浓缩，得到纯化的重组HSP70和meAg5蛋白；
将纯化的重组HSP70和meAg5蛋白和弗氏佐剂按照体积比1：1：2比例混合，混合后使用。
2.根据权利要求1所述的一种绵羊肺炎支原体亚单位疫苗的制备方法，其特征在于：从大肠杆菌(DE3)的裂解物中分离并纯化获得高纯度的重组蛋白，并与弗氏佐剂结合乳化制备亚单位疫苗。
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