| 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RT PCR检测猪IFN-α mRNA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501 bp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-α mRNA在诱导16 h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19 ku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8 g/L时，猪IFN-α成熟蛋白的表达量达到最大，为1.3 μg/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。 |
| 关键词: 猪α-干扰素 干酪乳酸杆菌 电穿孔转化 抗病毒 |
| DOI： |
| 分类号: |
| 基金项目:河南省重大科技专项（111100110300） |
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| Expression and reactivity of porcine interferon-alpha in Lactobacillus casei |
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MA Shi-jie,LI Kun,FU Peng-fei,et al
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| Abstract: |
| 【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-α gene was amplified from plasmid pMD18-IFN-α,and then directionally cloned into the down stream of lactose inducible promoter of integrative vector pMJ67.The integrative plasmid pMJ67-IFN-α 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 mRNA 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. |
| Key words: porcine interferon-alpha Lactobacillus casei electroporation antiviral |
