新城疫病毒F蛋白与鸡传染性支气管炎病毒S1蛋白的共表达、纯化及免疫原性

Co-expression,purification and immunogenicity of Newcattle disease virus F protein and chick infectious bronchitis virus S1 protein

目的共表达新城疫病毒(Newcastle disease virus,NDV)F蛋白与鸡传染性支气管炎(infectious bronchitis virus,IBV)S1蛋白,纯化并分析其免疫原性。方法根据NCBI中NDV F及IBV S1片段基因序列,截取包含F及S1蛋白部分抗原表位基因片段,设计并合成引物,PCR扩增目的片段,依次连接至原核表达载体pET-32a(+)上,构建重组质粒pET-32a-F-S1,转化至大肠埃希菌BL21(DE3)感受态细胞中,IPTG诱导表达,并对IPTG浓度和诱导时间进行优化,SDS-PAGE分析目的蛋白可溶性;重组蛋白经Ni-NTA agarose层析纯化复性后,Western blot法检测其反应原性。将14日龄雏鸡随机分F-S1、NDV F、IBV S1及PBS组,肌内免疫3轮,间隔10 d,每轮连续免疫5 d,每日1次,100μg/只,首次免疫后每隔7 d采集血清,ELISA法检测雏鸡血清特异性IgG水平。结果经双酶切鉴定,重组质粒p ET-32a-F-S1构建正确。重组蛋白最适IPTG诱导浓度为0.2 mmol/L,时间为3 h;其以包涵体形式存在,相对分子质量约为53000;纯化的重组蛋白能与抗NDV及抗IBV阳性血清反应,浓度为1.04 g/L。雏鸡免疫7~49 d,与PBS组比较,F-S1、NDV F及IBV S1组血清IgG水平均显著升高(P<0.01)。结论成功制备了重组蛋白F-S1,该蛋白具有良好的反应原性,能诱导雏鸡机体产生体液免疫应答,为NDV F及IBV S1重组蛋白的后续应用奠定了基础。

Objective To co-express,purify and analyze the immunogenicity of F protein of Newcasttle disease virus(NDV)and S1 protein of chick infectious bronchitis virus(IBV).Methods The gene fragments containing partial F and S1 antigen epitopes were selected according to the NDV F and IBV S1 gene fragments in NCBI,based on which primers were designed and synthesized for amplification of target gene fragments by PCR.The PCR products were inserted into prokaryotic expression vector pET-32 a(+).The constructed recombinant plasmids were transformed to competent E.coli BL21(DE3)in turn for expression under induction of IPTG.The IPTG concentration and time for induction were optimized.The solubility of target protein was analyzed by SDS-PAGE.The recombinant protein was purified by Ni-NTA agarose chromatography and refolded,and determined for reactogenicity by Western blot.The chicks at age of 14 d were divided into four groups,10 for each,and received three cycles of immunization with F-S1,NDV F,IBV S1 and PBS by intramuscular injection respectively,each at an interval of 10 d.Each cycle consisted of five doses,100μg for each,once a day for 5 d.Serum samples were collected every 7 d after the first injection and determined for specific IgG level by ELISA.Results Restriction analysis showed that recombinant plasmid pET-32 a-F-S1 was constructed correctly.The optimal IPTG concentration for induction was 0.2 mmol/L,while the optimal time was 3 h.The expressed recombinant protein,with a relative molecular mass of about 53000,existed in a form of inclusion body at a concentration of 1.04 g/L,which showed reaction with anti-NDV and anti-IBV positive sera.The serum IgG levels of chicks 7~49 d after the first injection with F-S1,NDV F and IBV S1 were significantly higher than that in PBS control group(P<0.01).Conclusion Recombinant F-S1 protein was successfully prepared,which showed good reactogenicity and induced humoral immune response in chicks.It laid a foundation of further application of recombinant NDV F and IBV S1 proteins.