摘要
目的重组HCV核心区和NS4区抗原．并原核表达该蛋白作为第四代HCV抗体检测试剂的补充抗原。方法用BioSun3．0软件分析HCV核心区和NS4区的抗原表位，选择具有优势抗原表位的3段多肽；然后用该3段多肽对应的基因设计合成引物，并采用RT—PCR和重叠延伸PCR技术扩增和拼接该3段基因；将拼接好的基因插入到原核表达载体pBVIL中，转化大肠埃希氏茵Topl0进行温度诱导表达；对表达的蛋白先后进行DEAE柱和分子筛柱纯化．复性后用问接ELlSA法分析该蛋白的免疫活性和作为补充抗原的可能性。结果通过RT—PCR和重叠延伸PCR技术获得了嵌合基因C-Ns4；用该基因构建的原核表达载体能表达融合蛋白C-NS4；融合蛋白C_Ns4经纯化后纯度良好，经复性后具有良好的HCV抗原性；并且用该蛋白包被酶联板，可以检测到现有第四代HCV抗体检测试剂漏检的样本。结论成功地重组了HCV核心区和NS4区抗原，原核表达了可以作为第四代HCV抗体检测试剂补充抗原的融合蛋白C—Ns4。
Objective To recompose HCV core and NS4 antigens,and prokaryoticly express the fusion protein used as com- plementary antigen of the fourth generation HCV-Ab detection reagent. Methods To analyze the antigenic epitopes of HCV core and NS4 domains by BioSun3.0, and select three fragments polypeptide comprising preponderant antigenic determi- nants. Then, the primers were designed and synthesized by using corresponding genes of the three fragments polypeptide,and the genes were amplified and spliced by reverse transcription PCR and overlap extension PCR. The spliced gene was inserted into prokaryotic expression vector pBVIL,which was to be transformed E. coil Topl0 for temperature induced expression. The expressed fusion protein was purified by DEAE column and molecular sieve column in turn. After being renatured,its immunocompetence was detected by indirect ELISA,and its feasibility used as complementary antigen was analyzed. Results The chimeric gene C-NS4 was attained by RT-PCR and overlap extension PCR. The prokaryotic expression vector eon- strutted by the gene could express fusion protein C-NS4. The fusion protein C-NS4 took on good purity after being purified and nicer HCV antigenicity after being renatured. And what＇s more, assay plate coated by the protein could detect the speci- mens that had been not detected by the fourth generation HCV-Ab detection reagent. Conclusion HCV core and NS4 anti- gens were successfully recompose＆ And the fusion protein C-NS4, which could be used as complementary antigen of the fourth generation HCV-Ab detection reagent,was prokaryoticly expressed.
出处
《现代检验医学杂志》
CAS
2012年第6期57-60,共4页
Journal of Modern Laboratory Medicine
关键词
丙型肝炎病毒
重组
表达
抗原性
hepatitis C virus
recombination
expression
antigenicity
