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目的 获得汉坦病毒重组核蛋白 ,并将其应用于血清学诊断。方法 采用PCR方法 ,从带有HTN型Z10株病毒全长核蛋白基因的质粒上扩增读码框的前 35 4bp的核蛋白基因片段。将截短的核蛋白基因片段插入表达载体pGEX 2 0T ,得到重组质粒pGEX2 0 Z10trNP ,转化大肠杆菌BL2 1(DE3) ,经IPTG诱导表达 ,表达产物经Glu tathioneSepharose 4B柱纯化 ,并进行抗原性及抗原特异性检测。结果 pGEX2 0 Z10trBP表达产物是相对分子质量约为 4 0 0 0 0的谷光甘肽转移酶 (GST)融合蛋白GST Z10trNP。经Westernblot分析 ,GST Z10trNP具有良好的抗原性。用纯化GST Z10trNP为抗原 ,用ELISA间接法检测出血热病人血清、出血热疫苗免疫的家兔及小鼠血清 ,均能很好地区分阴性和阳性血清。结论 GST Z10trNP表达量高 ,易于纯化 ,并且具有良好的抗原性及抗原特异性 ,是一种安全、廉价的诊断抗原。
Objective To obtain the recombinant nucleoprotein of Hantavirus and apply it to serological diagnosis. Methods The first 35 bp fragment of nucleoprotein of the reading frame was amplified by PCR from the plasmid carrying the full-length nucleoprotein of HTN Z10 strain. The truncated nucleoprotein gene fragment was inserted into the expression vector pGEX20T to obtain the recombinant plasmid pGEX20 Z10trNP, which was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was purified by Glu tathione Sepharose 4B column and subjected to antigenicity Antigen specific detection. Results The expression product of pGEX20 Z10trBP was glutathione transferase (GST) fusion protein GST Z10trNP with a relative molecular mass of about 40000. After Westernblot analysis, GST Z10trNP has good antigenicity. Using purified GST Z10trNP as antigen, ELISA indirect detection of hemorrhagic fever patients serum, hemorrhagic fever vaccine immunized rabbits and mice serum, can distinguish between negative and positive serum. Conclusion The expression of GST Z10trNP is high, easy to purify, and has good antigenicity and antigen specificity. It is a safe and inexpensive diagnostic antigen.