猪链球菌2型溶血素sly基因的表达及其产物的纯化和活性研究

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.

Study on expression of Streptococcus suis serotype 2 sly gene, purification and activity of its product

OBJECTIVE: To clone and express Streptococcus suis serotype 2 (S. suis 2) sly gene for constructing an foundation on identification of S. suis 2 protective antigen. METHODS: The sly gene was amplified from S. suis 2 clinical isolate strain 05ZYH33 genome DNA by PCR. The gene fragment was inserted into the expression vector pET-30b(+) to build pET30b-sly. When recombinant vector pET30b-sly was identified by restriction enzyme cutting and DNA sequencing as a correct one, subsequently it was transformed to E. coli Rosetta for expression under IPTG induction. The obtained fusion protein was purified by Ni-NTA affinity chromatography. The immunologic and hemolysis activity of the purified protein was proved through Western blot and hemolysis assay respectively. RESULTS: The PCR product was around 1500 bp. The gene segment inserted into the recombinant vector was proven to be completely identical with the sly gene sequence in the total genome sequence of S. suis 2. The target protein expressed was up to 30% of the total somatic protein under IPTG induction. The protein purity reached above 80% after purification. The protein could be recognized by human serum infected with S. suis 2 and could dissolve swine erythrocytes with the Hemolytic titer as 256. CONCLUSION: The expression vector pET30b-sly was successfully constructed. The target protein could be over-expressed in E. coli and possessed its biological activity after purification.