病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定

Prokaryotic expression and functional verification of ScFv antibody against viral hemorrhagic septicemia virus

为制备病毒性出血性败血症病毒(VHSV)单链抗体(single chain variable fragment antibody,ScFv)并鉴定其生物学功能,本研究提取抗VHSV单抗1G5的杂交瘤细胞株总RNA并反转录获得cDNA模板,通过PCR扩增VHSV抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其拼接成单链抗体Sc Fv基因后插入载体pET28a中,构建原核表达重组质粒并在大肠杆菌中诱导表达。结果表明,单链抗体主要以可溶性形式表达,分子量约28 ku,能特异性识别VHSV病毒的G蛋白并对VHSV病毒具有体外中和活性,其对VHSV病毒的G蛋白亲和力(KD)达到1.4×10^(–8) M。单链抗体ScFv的制备为进一步研究VHSV的治疗性抗体、快速诊断试剂奠定了基础。

The aim of this study is to prepare the ScFv antibody （single chain variable fragment antibody） against viral hemorrhagic septicemia virus （VHSV） and analyze the biological properties. The variable heavy （VH） and the variable light （VL）chain gene fragments were derived from mAb 1G5 hybridoma cells against VHSV. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in the pET28a vector to express ScFv protein in E.coli cells. The expressed ScFv protein, with a relative molecular mass of about 28 ku, existed in a form of soluble expression in cytoplasma. The ScFv protein could specifically identify VHSV glycoprotein （G）, and neutralize viral virulence of VHSV in vitro. The ScFv protein showed good affinity for VHSV glycoprotein （G） antigen, as indicated by KD values of 1.4× 10^-8 M. ScFv protein preparation has laid a foundation for further study of VHSV therapeutic antibodies as well as rapid diagnostic reagent.