Sip蛋白高亲和性噬菌体介导的牛源无乳链球菌间接ELISA方法的建立

发布时间：2018-04-23 09:16

  本文选题：无乳链球菌 + Sip蛋白　； 参考：《东北农业大学》2015年硕士论文

【摘要】：奶牛乳腺炎是奶牛最常见的疾病之一,影响奶牛的产奶量和牛奶品质,严重的可导致奶牛泌乳功能丧失甚至被迫淘汰,大大降低奶牛养殖的经济效益,给养牛业造成巨大的经济损失。同时病原微生物可通过污染的奶制品而感染人,对人类健康造成危害。无乳链球菌是奶牛乳腺炎的主要病原之一,无乳链球菌感染虽不引起乳房明显的脓肿或纤维化,但是可以导致腺体感染,造成产奶量持续减少。其表面免疫相关蛋白(Sip)暴露在细菌的表面,在多种血清型的无乳链球菌菌株中均有表达,是细菌重要的黏附和定植因子之一,具有较好的免疫保护作用和良好的抗原性,是无乳链球菌重要的候选疫苗抗原之一。九种不同血清型菌株Sip蛋白氨基酸序列比较结果显示此蛋白质高度保守。因此,利用此蛋白开发一个迅速有效的检测方法监控无乳链球菌感染,提高乳制品的质量和安全,具有重要的应用价值。本研究参照Gen Bank公布的牛源无乳链球菌Sip基因序列(AF151361)设计一对特异性引物,成功构建p GEX-6p-1-Sip原核表达质粒。经IPTG诱导表达及条件优化,成功在大肠杆菌感受态Rosetta(DE)中表达了分子量为59.8 Ku的无乳链球菌Sip重组蛋白,其优化条件为终浓度1.0 mmol/L IPTG 37℃恒温诱导5 h。纯化的重组蛋白为其后噬菌体的筛选提供了材料。随后,以纯化的无乳链球菌Sip重组蛋白为靶分子,用M13噬菌体c DNA文库,进行4轮噬菌体生物淘选。无乳链球菌Sip蛋白的首轮包被量为10μg/孔,此后每轮依次减半,噬菌体每轮投入量均为1.5×1011 CFU/μL。经过噬菌体淘洗、扩增和滴度测定,最终在总数少于100的LB/IPTG/Xgal平板上随机挑取10个噬菌体蓝斑于摇菌管中进行4.5 h扩增后测定其滴度。扩增后的10个噬菌体分别进行噬菌体与靶分子的噬菌体ELISA亲和能力的测试,检测结果显示,所获取的10个随机噬菌体均与靶分子具有高度的亲和能力,为ELISA方法检测无乳链球菌的建立奠定了基础。利用能与无乳链球菌Sip蛋白结合的噬菌体作为第一抗体,M13多抗作为第二抗体,建立了可快速检测含无乳链球菌奶样的间接ELISA检测方法并进行了条件优化。优化结果为:抗原包被浓度(即敏感度)为10 CFU/mL/孔,噬菌体最佳作用浓度为108 PFU/mL;抗原最佳包被方式确定为37℃包被2 h时后4℃过夜的方式;最佳封闭剂的选择为1.0%BSA磷酸盐缓冲液置37℃封闭3 h;噬菌体最佳孵育时间为60 min;M13多抗1:1000倍稀释孵育45 min;酶标抗体最佳稀释度为1:4000,最佳孵育时间为45 min。并用所建立的间接ELISA检测方法进行了无乳链球菌敏感性、特异性和临床样本的检测,敏感性可检测到10 CFU/mL。临床样本检测了某牧场54份患奶牛乳腺炎的奶样,检测阳性率达14.286%。本实验将噬菌体表面展示技术与ELISA检测方法相结合,为奶牛乳腺炎无乳链球菌提供了简便高效的新型检测方法。
[Abstract]:Cow mastitis is one of the most common diseases in dairy cows, which affects the milk yield and quality of cows, which can seriously lead to the loss of lactation function and even forced elimination of dairy cows, which greatly reduces the economic benefits of dairy cattle breeding. Great economic losses have been caused to the cattle industry. At the same time, pathogenic microorganisms can infect people through contaminated dairy products, which is harmful to human health. Streptococcus actinomycetes is one of the main pathogens of dairy cow mastitis. Although it does not cause abscess or fibrosis of breast, it can cause gland infection and decrease milk production. The surface immune-associated protein (Sip-Sip) is expressed on the surface of bacteria and expressed in various serotypes of Streptococcus lactis. It is one of the important adhesion and colonization factors of bacteria, and has good immune protection and antigenicity. It is one of the most important candidate vaccine antigens for Streptococcus lactobacillus. The amino acid sequence analysis of Sip protein of nine different serotype strains showed that the protein was highly conserved. Therefore, it is of great value to develop a rapid and effective method to detect streptococcus lactococcus infection and improve the quality and safety of dairy products. In this study, a pair of specific primers were designed according to the Sip gene sequence AF151361 published by Gen Bank, and the prokaryotic expression plasmid of p GEX-6p-1-Sip was successfully constructed. The recombinant protein of Streptococcus lactis Sip with molecular weight of 59.8 Ku was successfully expressed in Escherichia coli (E. coli) by IPTG induced expression and optimized conditions. The optimal conditions were as follows: 1. 0 mmol/L IPTG at 37 鈩，

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