菜豆荚斑驳病毒外壳蛋白大小亚基的原核表达及其抗血清制备

Prokaryotic expression of the large and small subunit gene of the Bean pod mottle virus coat protein and their antiserum preparation

本研究对菜豆荚斑驳病毒大、小亚基编码的基因密码子进行了优化并人工合成了这两个基因,然后克隆到原核表达载体p ET-22b（＋）中,通过转化大肠杆菌BL21（DE3）菌种并在IPTG的诱导下进行了成功表达,大亚基表达产物分子量为41 k D、小亚基表达产物分子量为22 k D,并制备出了大小亚基基因表达产物的抗血清。测试结果表明,优化后的CP的大、小亚基基因在37℃、1.0 mmol/L IPTG诱导下,3 h后得到了成功的表达,制备的两种抗血清特异性强、效价都达到1∶3.2×10~4,可以对BPMV CP 3个不同的区域同时进行检测,提高了检测的准确度。

The large subunit（L） and small subunit（S） gene of the Bean pod mottle virus （BPMV） coat protein （CP）were artificially synthesized by amino acids codon optimied and then cloned into prokaryotic expression vector pET-22b （＋） respectively and finally transformed into E. coli BL21 （DE3） strain. The results of SDS-PAGE showed that 41 kD L and 22 kD S were expressed effectively when induced with IPTG at 37℃ for three hours. The antisera with high specificity were obtained when the rabbits were immunized with the purified expressed proteins and the titer of the both antisera was 1：3.2×10^4 assayed by indirect ELISA,respectively. The test results will be more accurate because the three different domains of the CP can be inspected at the same time.