中蜂囊状幼虫病毒VP1基因的优化表达及其免疫原性分析  

Expression and immunogenicity of optimized Chinese sacbrood virus VP1 protein

作　　者：张皓淳 宋立先 万红霞 马鸣潇[1] 曲祖乙[1] 费东亮[1] 钟义[1] 

机构地区：[1]辽宁医学院畜牧兽医学院,辽宁锦州121000 [2]山东省庆云县人民医院内科,山东庆云253700 [3]内蒙古赤峰市翁牛特旗医院,内蒙古赤峰024500

出　　处：《中国生物制品学杂志》2015年第5期468-474,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31372435);辽宁省科技厅自然基金(联合基金)(2014022047)

摘　　要：目的原核表达优化的中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)的VP1基因,并分析其免疫原性。方法根据大肠埃希菌密码子偏爱性对野生型CSBV VP1(wt VP1)基因进行优化(opti VP1),将基因wt VP1和opti VP1分别克隆至p GEX-6P-1原核表达系统,构建原核表达质粒p GEX-6P-wt VP1和p GEX-6P-opti VP1,分别转化E.coli BL21,经IPTG诱导表达,并对诱导温度、IPTG终浓度及诱导时间进行优化,诱导蛋白经Western blot鉴定;重组蛋白经GSH-琼脂糖凝胶层析纯化;分别用PBS、纯化的GST标签蛋白、纯化的重组蛋白及CSBV纯化病毒免疫小鼠,经间接ELISA法检验各组小鼠的血清抗体效价。结果优化后的VP1基因序列降低了密码子影响指数,提高了密码子适应指数;质粒p GEX-6P-wt VP1和p GEX-6P-opti VP1经鉴定证明构建正确;最佳诱导表达条件为:IPTG终浓度0.8 mmol/L,30℃,220 r/min诱导8 h;诱导表达蛋白占菌体总蛋白百分比由wt VP1菌株(5.49±0.30)%提高至opti VP1菌株(59.7±0.51)%,纯化后纯度可达0.5 mg/ml;兔抗GST标签抗体及兔抗CSBV血清均与优化后的重组蛋白发生特异性反应;重组蛋白组小鼠二免后可产生较高水平抗体,明显高于PBS组和GST标签蛋白组(P<0.05)。结论通过密码子优化实现了CSBV结构基因VP1的高效表达,表达蛋白可诱导小鼠机体产生特异性抗体,具有良好的免疫原性,为探讨CSBV感染的分子发病机制和制备抗CSBV病毒的多克隆抗体奠定了基础。Objective To express the VP1 gene of Chinese sacbrood virus（CSBV） in prokaryotic cells and analyze the immunogenicity of expressed product. Methods Based on the E. coli codon preference, wild-type CSBV VP1（wt VP1）gene was optimized to obtain opti VP1 gene. The wt VP1 and opti VP1 genes were cloned into prokaryotic expression vector p GEX-6P-1 respectively, and the constructed recombinant plasmids p GEX-6P-wt VP1 and p GEX-6P-opti VP1 were transformed to E. coli BL21 and induced with IPTG. The temperature and time for induction as well as the final concentration of IPTG were optimized. The expressed protein was identified by Western blot and purified by GSH-agarose gel chromatography. Mice were immunized with PBS, purified GST tag, purified recombinant protein and purified CSBV respectively, and determined for serum antibody titer by indirect ELISA. Results The influencing index of codon of Opti VP1 gene decreased, while the adaptation index increased. Plasmids p GEX-6P-wt VP1 and p GEX-6P-opti VP1 were constructed correctly. The condition for expression was optimized as induction with IPTG at a final concentration of 0. 8 mmol / L at30 ℃, 220 r / min for 8 h. The expression level of wt VP1 was（5. 49 ± 0. 30）%, while that of opti VP1 increased to（59. 7 ±0. 51）%, of total somatic protein. The purity of opt VP1 was 0. 5 mg / ml after purification. Both rabbit anti-GST tag antibody and rabbit antisera against CSBV showed specific reactions with the optimized recombinant protein. The antibody level of mice immunized twice with the recombinant protein was significantly higher than those with PBS and with GST tag（P 0. 05）. Conclusion CSBV VP1 structural gene was highly expressed in a fusion form by codon optimization, and expressed opti VP1 induced specific antibody in mice and showed good immunogenicity, which laid a foundation of re-search on molecular mechanism of pathogenesis and preparation of polyclonal antibody against CSBV.

关 键 词：中蜂囊状幼虫病毒 VP1结构蛋白 原核表达 免疫原性 

分 类 号：R373.24[医药卫生—病原生物学] R392-33[医药卫生—基础医学]