论文部分内容阅读
目的克隆并表达猪链球菌2型(SS2)溶血素基因(sly),为筛选SS2疫苗保护性抗原奠定基础。方法用PCR方法从SS2临床分离株05ZYH33的基因组DNA中扩增出溶血素基因片段,将目的基因插入表达载体pET-30b(+)中,构建重组表达载体pET30b-sly。重组载体经限制性内切酶酶切和DNA测序鉴定后,转化大肠埃希菌(E.coli Rosetta)。IPTG诱导表达,镍离子亲和层析纯化重组蛋白,鉴定目的蛋白的免疫学活性以及溶血活性。结果PCR扩增的sly基因长度约为1500 bp,经测序分析,插入载体的sly基因序列准确并保持了正确的读框。经IPTG诱导的目的蛋白表达量约占总蛋白的30%,亲和层析纯化后,蛋白纯度达80%以上。Western blot检测证实该蛋白能与感染SS2的人血清发生特异性结合。溶血试验表明重组蛋白溶血素能使猪红细胞发生溶解,溶血价为256。结论成功构建了表达载体pET30b-sly,该载体可在大肠埃希菌中表达,表达蛋白具有免疫反应原性及溶血活性。
Objective To clone and express the sly gene of Streptococcus suis type 2 (SS2) and lay the foundation for the screening of the protective antigen of SS2 vaccine. Methods The hemolysin gene fragment was amplified from genomic DNA of SSZ clinical isolate 05ZYH33 by PCR. The target gene was inserted into the expression vector pET-30b (+) to construct the recombinant expression vector pET30b-sly. After restriction endonuclease digestion and DNA sequencing, the recombinant vector was transformed into E. coli Rosetta. IPTG induced expression, purification of recombinant protein by nickel ion affinity chromatography, identification of the target protein immunological activity and hemolytic activity. Results The length of sly gene amplified by PCR was about 1500 bp. After sequencing, the sly gene sequence inserted into the vector was accurate and maintained the correct reading frame. The target protein induced by IPTG accounted for about 30% of the total protein, purified by affinity chromatography, the protein purity of more than 80%. Western blot analysis confirmed that the protein could specifically bind to human SS2-infected human serum. Hemolysis test showed that the recombinant protein hemolysin can make pig erythrocytes lytic, hemolysis price of 256. Conclusion The expression vector pET30b-sly was successfully constructed. The vector was expressed in Escherichia coli. The expressed protein was immunogenic and hemololytic.