李斯特菌载体结核疫苗候选株基因重组构建及蛋白表达验证

Genetic Recombiniation and Protein Expression Detection of Listeria-based Tuberculosis Vaccine Candidates

目的基因重组构建以单增李斯特菌、绵羊李斯特菌为载体的新型结核疫苗候选株,并进行蛋白表达验证,为结核疫苗研究和结核防控提供新思路和新方法。方法以体外基因连接、质粒转化等技术,将本实验室已有的分别编码结核抗原Ag85C、ESAT-6蛋白的两种基因盒,插入携带单增李斯特菌、绵羊李斯特菌同源序列的打靶质粒中。将打靶质粒电转化单增李斯特菌、绵羊李斯特菌,在42℃和30℃连续传代,利用同源重组杂交原理和基因打靶技术,将打靶质粒携带的两种编码结核抗原的抗原基因盒,分别整合至致病基因(actA和plcB)被敲除的单增李斯特菌、绵羊李斯特菌减毒株及未经减毒的绵羊李斯特菌基因组中。重组菌经蓝白斑、抗生素抗性及PCR筛选,再经Western blot检测其菌体蛋白及分泌蛋白的表达情况。结果构建了携带抗原基因盒的重组菌株5株,其重组基因序列PCR验证结果均符合预期,且测序验证成功;重组菌不携带红霉素抗性基因,表型特征符合预期;其中,重组菌Li-Rv0129c、Li-ΔactAplcB-Rv0129c按预期表达了结核杆菌Ag85C抗原蛋白,Li-ΔactAplcBRv3875按预期表达了结核杆菌ESAT-6抗原蛋白,Lm重组菌未表达相应结核抗原蛋白。结论成功构建并筛选得到3株以绵羊李斯特菌为载体的、携带结核杆菌Rv0129c(编码Ag85C)抗原基因盒或Rv3875(编码ESAT-6)抗原基因盒,且表达相应抗原蛋白的新型结核疫苗候选株。

Objective Genetic construction of tuberculosis vaccine candidates based on Listeria （L.）monocytogenes, L. ivanovii, and evaluation their protein expression, in order to provide a novel method for researchon tuberculosis controlling. Methods Two kinds of gene cassettes carrying tuberculosis antigen encoding gene Rv3875 or Rv0129c were inserted into targeting vector harboring L. monocytogenes, L. ivanovii homologous sequences via genetic connection methods and plasmid transformation technology in vitro. Targeting plasmids were electroporated into L. monocytogenes, L. ivanovfi, and the recombinant strains were experienced serial passage at 42 ℃ and 30 ℃. Subsequently, the tuberculosis antigen gene cassettes in targeting plasmids were integrated into L.monocytogenes and L. ivanovii attenuated strain （knocking out of virulence gene actA and plcB） and L. ivanovii wild type strain by homologous recombination and gene targeting technology. The recombinant strains were screenedby blue-white spot and antibiotic resistance test; the intracellular and extracellular proteins of the recombinant strains were tested by Western blot. Results Five recombination strains carried antigen gene cassette were .constructed, and the recombinant genome were confirmed by PCR and sequencing. No erythromycin resistance gene was found in 5 strains, which Was coincident to expection. Recombination strains Li-Rv0129c, Li-△actAplcB-Rv0129c and Li-△actAplcB-Rv3875 expressed Mycobacterium tuberculosis antigenic protein, Ag85C or ESAT-6, as expected. But L. monocytogenes strains did not express proper antigenic protein. Conclusion Three novel L.ivanovii-based tuberculosis vaccine candicates, carrying Mycobacterium tuberculosis RvO129c antigen gene cassette （coding for Ag85C） or Rv3875 gene cassette （coding for ESAT-6）, and expressing relevant antigenic proteins have been successfully selected.