猪带绦虫TSOL18重组乳球菌分泌型和非分泌型疫苗构建及鉴定  

Construction and identification of TSOL18 recombinant Lactococcus lactis secretory and non-secretory vaccines of Taenia solium

作　　者：孙俊超[1] 李想 周必英[1] Sun Junchao;Li Xiang;Zhou Biying(Department of Parasitology,Zunyi Medical College,Zunyi 563000,Chin)

机构地区：[1]遵义医学院寄生虫学教研室,遵义563000

出　　处：《中华地方病学杂志》2018年第8期603-606,共4页Chinese Journal of Endemiology

基　　金：贵州省优秀青年科技人才基金（黔科合平台人才[2017]5629）;贵州省卫计委科研基金（gzwjkj2016-1-048）

摘　　要：目的构建猪带绦虫重组非分泌型pMG36e-TSOL18／L．lactis和分泌型pMG36e-SP-TSOL18／L．1actis疫苗。方法以GenBank中登录的猪带绦虫TSOL18基因序列为模板，以乳酸菌为宿主系统进行基因优化，采用基于PCR的精确合成（PAS）方法，设计引物，在其两端分别加入酶切位点Sac I/HindⅢ以及保护性碱基，在其N端添加SPUSP45分泌信号肽序列，分别合成TSOL18和SP-TSOL18目的基因。将目的基因克隆至大肠埃希菌-乳球菌穿梭表达质粒pMG36e，构建胞内型表达载体pMG36e-TSOL18和分泌型表达载体pMG36e-SP-TSOL18．进行测序和酶切鉴定；采用电穿孔转化法将两种重组质粒转化至乳酸乳球菌（Llactis）MG1363，构建猪带绦虫重组pMG36e-TSOL1／L．lactis和pMG36e-SP-TSOL18／L．lactis疫苗，并进行PCR鉴定。结果经SacI和HindⅢ双酶切．获得了相应的TSOL18基因片段和pMG36e载体片段，与预期结果相符；与TSOL18基因标准序列作比对，匹配度均为100％，均插入到pMG36e载体的SacI和HindⅢ中。重组pMG36e-TSOL18／L．lactis和pMG36e-SP-TSOL18／L．lactis的PCR结果均可见393bp的基因片段产物。结论成功构建了猪带绦虫重组pMG36e-TSOL18／L.lactis和pMG36e-SP-TSOL18／L．lactis疫苗。Objective To construct recombinant non-secretory pMG36e-TSOL18/L.lactis and secretory pMG36e-SP-TSOL18/L.lactis vaccines of Taenia solium. Methods Taking sequence of Taenia solium TSOL18 gene as template, genetic optimization was carried out using lactic acid bacteria as a host system, TSOL18 gene and SP-TSOL18 gene were synthesized using PCR-based accurate synthesis （PAS）, through the design of full-length primers, the addition of restriction enzyme cutting sites Sac I and Hind Ⅲ and protective bases, and the SPUSP45 secretory signal peptide sequence in the N terminal. TSOL18 gene and SP-TSOL18 gene were cloned into Escherichia coli-L.lactis shuttle expression plasmid pMG36e to construct intracellular expression vector pMG36e- TSOL18 and secreted expression vector pMG36e-SP-TSOL18. The two recombinant plasmids were identified by enzyme digestion and sequencing, and electroporated into L.lactis MG1363 to construct the recombinant pMG36e- TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis vaccines of Taenia solium, and they were identified by PCR. Results TSOL18 gene fragment and pMG36e vector fragment were obtained by double digestion with Sac I and Hind lll, which were consistent with the expected results; TSOL18 gene standard sequence was aligned and the matching degree was 100%, and both were inserted into Sac I and Hind Ⅲ of pMG36e vector. Our PCR results showed that both recombinant pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis were 393 bp gene fragment products. Conclusion The recombinant pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis vaccines of Tacnia solium are successfully constructed.

关 键 词：猪带绦虫 DNA 重组 乳球菌属 疫苗 鉴定 

分 类 号：R392[医药卫生—免疫学]