摘要
为构建抗PRRSV与抗人红细胞单链抗体(2E8-Sc Fv)融合的双功能单链抗体基因并预测其结构和功能,本研究以PRRSV杂交瘤细胞株为模板,通过RT-PCR和SOE-PCR构建PRRSV-Sc Fv基因,将其重组到2E8-Sc Fv C端构建双功能单链抗体基因并测序分析,利用生物信息学软件预测其结构功能。结果显示,成功获得长735 bp的PRRSV-Sc Fv基因,由Linker、VH和VL组成,VH、VL均含明确的CDR和FR区及特征性Cys,与多种鼠源Sc Fv基因高度同源,具有重组功能性鼠源抗体可变区特征,与2E8融合后形成全长1 470 bp的2E8-PRRSV-Sc Fv基因。经生物信息学分析,融合基因编码490个氨基酸,亲水性评估值为-0.365,为亲水性蛋白;结构预测分析显示,其存在丰富的二级结构,可折叠形成含有多个沟槽结构的空间构象,利于抗原的结合,理论上具有良好的抗原结合活性。本研究成功构建抗PRRSV双功能单链抗体基因并对其编码的蛋白质结构进行了预测,为今后进行融合蛋白的表达及最终研究PRRSV抗原快速检测方法奠定基础。
The purpose of this research is to construct a bifunctional single chain antibody gene 2E8-PRRSV-Sc Fv by fusing PRRSV antibody with 2E8-Sc Fv and to predict its structure and function. Firstly, PRRSV hybridoma cell strain was used as a mold to construct PRRSV-Sc Fv gene through RT-PCR and SOE-PCR, then this gene was cloned into the C terminal of single chain antibody of anti-human erythrocyte H antigen gene(2E8-Sc Fv) to construct a bifunctional single chain antibody gene, after which sequencing analysis was done and protein structure was predicted by using software for bioinformatics. The results showed that the PRRSV-Sc Fv gene was 735 bp,composed of 45 bp linker, 363 bp VH and 327 bp VL, in which the VH and VL both contained definite CDR zone,FR zone and characteristics of cysteine residues. Results also showed that the PRRSV-Sc Fv gene was highly homologous with mouse-borne single-chain genes, having features of functionally rearranged variable regions of mouse-borne antibodies. A 2E8-PRRSV-Sc Fv gene of 1 470 bp was formed after the fusion of PRRSV-Sc Fv and2E8. Bioinformatics analysis indicated that the fused gene encoded 490 amino acids, its hydrophilic value was-0.365, suggesting it was a hydrophilic protein; structure analysis showed that in the gene there were various second structures which could be folded to form a space conformation with multiple groove structures. All these suggested a favorable condition for antigen binding and theoretically good activities. In this study, an anti-PRRSV bifunctional single-chain antibody gene was successfully constructed and its protein structure was predicted. The data and results will provide foundation for subsequent expression of fusion protein and researches on quicker detection of PRRSV antigen.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2015年第5期988-994,共7页
Genomics and Applied Biology
基金
广西重点实验室建设项目(12-071-28-A-2)
广西自然基金项目(桂科自0991219)共同资助
