猪IFN-α基因在干酪乳酸杆菌中的表达及反应活性鉴定  

Expression and reactivity of porcine interferon-alpha in Lactobacillus casei

作　　者：马世杰[1] 李坤[1] 付朋飞[1] 郭晓庆[1] 高晓云[1] 崔保安[1] 陈红英[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《西北农林科技大学学报（自然科学版）》2015年第7期29-34,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：河南省重大科技专项(111100110300)

摘　　要：【目的】构建能够稳定表达猪α-干扰素(IFN-α)成熟蛋白的重组干酪乳酸杆菌(Lactobacillus casei CECT5276),为将其作为口服疫苗来提高动物机体免疫力奠定基础。【方法】从质粒pMD18-IFN-α扩增出猪IFN-α成熟蛋白基因,再将其定向克隆到干酪乳酸杆菌整合型表达载体pMJ67的lac启动子下游,得到重组质粒pMJ67-IFN-α,电穿孔转化干酪乳酸杆菌。抽提干酪乳酸杆菌基因组DNA,进行PCR扩增及测序鉴定。采用半定量RTPCR检测猪IFN-αmRNA在干酪乳酸杆菌中的转录水平;采用Western blot对重组干酪乳酸杆菌菌体进行分析,检测其表达产物的活性;用双抗体夹心ELISA法确定最适的乳糖诱导质量浓度。【结果】PCR扩增获得了501bp的IFN-α,成功构建了重组质粒pMJ67-IFN-α,转化干酪乳酸杆菌后获得了重组乳酸杆菌。半定量RT-PCR结果显示,IFN-αmRNA在诱导16h时表达水平最高;Western blot分析表明,构建的重组干酪乳酸杆菌成功表达出了相对分子质量约19ku的重组蛋白,其可与鼠抗猪IFN-α抗体发生特异性反应。ELISA结果表明,当乳糖诱导质量浓度达到8g/L时,猪IFN-α成熟蛋白的表达量达到最大,为1.3μg/L。【结论】将猪IFN-α成熟蛋白基因整合到干酪乳酸杆菌基因组中,构建的重组乳酸杆菌能够稳定表达猪IFN-α成熟蛋白。[Objective] This study aimed to construct the recombinant Lactobacillus casei CECT5276 that could stably express porcine IFN-α to increase the immunity of animals as an oral vaccine. [Method] Porcine IFN-a gene was amplified from plasmid pMD18-IFN-α, and then directionally cloned into the downstream of lactose-inducible promoter of integrative vector pMJ67. The integrative plasmid pMJ67-IFN-a was transformed into Lactobacillus casei CECT5276 by electroporation. Genomic DNA was extracted and identified by PCR product sequencing. The transcriptional level of porcine IFN-α rnRNA was detected in recombinant Lactobacillus casei CECT5276 by RT-PCR. The expression product of the recombinant Lactobacillus casei CECT5276 and concentration of lactose induction were detected by Western blot and ELISA.[Result] The recombinant Lactobacillus casei CECT5276 （501 bp） was identified by PCR product sequencing. The highest expression level of porcine IFN-α mRNA was detected 16 hours after induction in recombinant Lactobacillus casei CECT5276,as revealed by RT-PCR for amplification of porcine IFN-α gene. The 19 ku expressed protein of porcine IFN-α and the specificity were identified by Western blot. The optimal concentration of lactose induction of 8 g/L was tested by ELISA and the highest expression level was 1.3 μg/L. [Conclusion] The recombinant Lactobacillus casei was obtained successfully.

关 键 词：电力系统 网格化技术 应用 

分 类 号：S852.651[农业科学—基础兽医学]