猪传染性胃肠炎病毒spike蛋白亲和肽的原核表达

Prokaryotic expression of affinity peptides to spike protein of transmissible gastroenteritis virus

【目的】原核表达猪传染性胃肠炎病毒(transmissible gastroenteritis virus of swine,TGEV)spike蛋白亲和肽(SQHT),检测其病毒亲和性,为建立TGEV诊断方法奠定理论和物质基础。【方法】人工合成TGEV spike蛋白亲和肽基因,亚克隆后将该基因分别插入至原核表达载体pET-32a和pGEX-6p-1中,构建重组质粒pET-32a-SQHT和pGEX-6p-SQHT,对重组质粒进行BamHⅠ单酶切和BamHⅠ/XhoⅠ双酶切及测序鉴定。将重组质粒分别转化至大肠杆菌Rosetta(DE3),用IPTG进行诱导表达,对其表达产物的生物学活性进行检测。【结果】亚克隆获得了150 bp的TGEV spike蛋白亲和肽基因。成功构建了重组质粒pET-32a-SQHT和pGEX-6p-SQHT,并诱导表达出重组蛋白TRX-SQHT和GST-SQHT,其分子质量分别为25和31 ku。Western blot分析表明,2种重组蛋白与TGEV病毒粒子具有良好的亲和性;Dot-ELISA分析表明,2种重组蛋白与TGEV病毒粒子的最低结合滴度TCID50为5×102 mL-1。特异性试验表明,重组蛋白TRX-SQHT和GST-SQHT均可与TGEV产生特异性结合,而不与猪流行性腹泻病毒(PEDV)和猪轮状病毒(PoRV)结合。【结论】使用原核细胞成功表达了TGEV spike蛋白亲和肽,表达的重组蛋白具有很好的TGEV特异亲和性。

【Objective】 Prokaryotic expression of affinity peptides to spike protein of transmissible gastroenteritis virus(TGEV) and analysis of viral affinity activity were conducted to provide basis for establishment of TGEV diagnostic methods.【Method】 In this study,a tandem gene containing the TGEV spike protein affinity peptide was synthesized.After sub-cloning,the gene was inserted into prokaryotic expression vectors of pET-32 a and pGEX-6 p-1 to obtain recombinant plasmids pET-32 a-SQHT and pGEX-6 p-SQHT.The recombinant plasmids were identified by single enzyme digestion of BamHⅠ,double enzyme digestion of BamHⅠ/XhoⅠ and sequencing.Then,the recombinant plasmids were transformed into E. coli Rosetta(DE3) and induced by IPTG to obtain the expression of recombinant protein.Finally,the biological activity of expression products was measured.【Result】 A gene about 150 bp of TGEV spike protein affinity peptide was obtained by sub-cloning.Two recombinant plasmids, pET-32 a-SQHT and pGEX-6 p-SQHT,were constructed and expressed by induction successfully.The molecular weights of the two recombinant proteins were 25 and 31 ku,respectively.Western blot analysis showed that the two recombinant proteins had good affinity with TGEV virions.Dot-ELISA analysis showed that the minimum binding titer of the two recombinant proteins binding to the TGEV virions was TCID50 5×102 mL-1.Specificity experiments showed that the recombinant proteins TRX-SQHT and GST-SQHT did not bind to PEDV or PoRV.【Conclusion】 The affinity peptides to spike protein of TGEV were successfully expressed using prokaryotic cells and the two recombinant proteins had good specificity affinity to TGEV.