抗新城疫病毒HN蛋白单链抗体的原核表达及其功能鉴定

Prokaryotic expression and functional analysis of the single-chain antibody against the HN protein of Newcastle disease virus

为制备抗鸡新城疫病毒(NDV)血凝素-神经氨酸酶(HN)单链抗体(scFv)并鉴定其生物学功能,本研究提取抗NDV HN单克隆抗体杂交瘤细胞株总RNA,通过RT-PCR扩增抗HN蛋白抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其插入含有Linker序列的载体pET-scIG中,构建抗HN的scFv(HN-scFv)原核表达重组质粒pET-HN-scFv并在大肠杆菌中诱导表达。结果显示HN-scFv主要以包涵体形式存在。将包涵体进行复性和Ni-NTA层析柱纯化后用于流式细胞仪检测,结果显示复性纯化后的HN-scFv能够识别表达于293T细胞表面的NDV HN蛋白。该scFv的制备为进一步对该抗体进行改造奠定了基础。

To express the single-chain antibody against the HN protein （HN-scFv） of Newcastle disease virus （NDV） in bacteria and identification its specific binding activity, the total RNAs were extracted from a hybridoma producing monoclonal antibody （MAb） against HN of NDV. The variable regions encoding the heavy and light chains of MAb, named VH and VL respectively, were amplified by RT-PCR and inserted into pET-scIG vector to construct the recombinant plasmid of pET-HN-scFv. The recombinant plasmid was transformed into the E.coli （BL21） for HN-scFv expression. In addition, the expression of HN-scFv was induced and analyzed in SDS-PAGE assay, and the results showed that the expressed HN-scFv was mainly in the form of inclusion bodies. Moreover, the testing with flow cytometrey indicated that the renatured HN-scFv was able to specifically recognize the HN expressed on 293T cells. These results revealed that we successfully constructed and functionally expressed the HN-scFv in bacteria which would facilitate the engineering of the MAb in the future.