无乳链球菌表面蛋白重组制备及检测方法初步建立

发布时间：2018-05-01 21:19

本文选题：无乳链球菌 + 表面蛋白　；参考：《天津科技大学》2012年硕士论文

【摘要】：奶牛乳腺炎是世界奶牛业的主要危害因素之一,它不仅影响产奶量,造成经济损失,而且影响牛奶的品质,危害人类的健康。而无乳链球菌是引起奶牛乳腺炎的常见病原微生物之一,因而是乳业关心的主要问题。建立无乳链球菌快速检测方法,对于提高奶制品质量与安全,推进奶牛业发展,促进人类健康意义重大。本研究克隆表达了三种无乳链球菌表面蛋白(Rib,Sip, Alpha),并分别进行了抗体制备,为无乳链球菌快速检测试纸条和多价疫苗的研制奠定了良好的基础。本实验首先将6种血清型的ATCC无乳链球菌标准菌进行了活化,并分别提取了基因组DNA。根据Rib, Sip, Alpha三种表面蛋白基因序列设计特异性引物,以不同标准菌株基因组DNA为模板,分别扩增得到长度为453bp、1302bp和699bp的片段,将片段克隆连接至pMD18-T载体进行序列测定,结果表明获得的片段为正确的目的基因。将rib和sip基因片段亚克隆至重组表达载体pET26b、alpha基因片段亚克隆至重组表达载体pGEX-4T-1,并利用IPTG进行诱导表达,SDS-PAGE结果显示3种基因均能够重组表达,外源蛋白的分子量分别为18kDa、61kDa和52kDa,与预期大小相符,但是其中Rib和Alpha形成包涵体。将3种基因进行大规模重组表达并对形成的包涵体进行变性、复性后,利用亲和纯化获得了纯度较高的目的蛋白。经过质谱鉴定,3种外源蛋白与与无乳链球菌Rib,Sip以及Alpha的相似度分别达到99.99%,100%以及100%。将三种蛋白分别免疫新西兰白兔,制备多克隆抗体,经测定抗体效价分别为480000：1,640000：1以及320000：1。利用3种抗血清,分别采用间接ELISA方法对ATCC标准菌进行检测,结果显示3种多克隆抗体均体现出一定亲和性和特异性。
[Abstract]:Dairy cow mastitis is one of the main harmful factors of dairy industry in the world. It not only affects milk production, causes economic loss, but also affects the quality of milk and human health. Streptococcus actinomycetes is one of the common pathogenic microorganisms that cause dairy cow mastitis, so it is the main concern of dairy industry. It is of great significance to establish a rapid detection method for streptococcus lactococcus to improve the quality and safety of dairy products, promote the development of dairy industry and promote human health. In this study, three surface proteins of Streptococcus lactobacillus were cloned and expressed, and the antibodies were prepared, which laid a good foundation for the rapid detection of streptococcus lactobacillus and the development of multivalent vaccine. In this study, six serotypes of ATCC were first activated and genomic DNAA were extracted. According to the three surface protein gene sequences of Rib, Sip, Alpha, specific primers were designed and amplified by using genomic DNA of different standard strains as templates. The fragments of 453bpC1302bp and 699bp were amplified and cloned into pMD18-T vector for sequencing. The results showed that the obtained fragment was the correct target gene. The rib and sip gene fragments were subcloned into the recombinant expression vector pET26bGN alpha gene and subcloned into the recombinant expression vector pGEX-4T-1. The results of SDS-PAGE showed that all the three genes could be expressed by SDS-PAGE. The molecular weights of exogenous proteins were 18kDa 61kDa and 52kDa respectively, which were consistent with the expected size, but Rib and Alpha formed inclusion bodies. The three genes were expressed in large scale and the inclusion bodies were denatured. After renaturation, the purified target protein was obtained by affinity purification. The similarity of three exogenous proteins with Streptococcus lactis Ribsip and Alpha was 99.9999% and 100%, respectively. The polyclonal antibodies were prepared by immunizing New Zealand white rabbits with three proteins. The titers of the antibodies were 480000: 1, 6400: 1 and 320 000: 1, respectively. Three kinds of antiserum were used to detect ATCC standard bacteria by indirect ELISA method. The results showed that the three polyclonal antibodies showed certain affinity and specificity.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.57

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