中国兽医科学2025，Vol.55Issue(4)：463-469,7.DOI:10.16656/j.issn.1673-4696.2025.0052
旋毛虫乳酸脱氢酶基因的克隆表达及组织定位
Cloning,expression and localization of lactate dehydrogenase gene of Trichinella spiralis
摘要
Abstract
In order to study the reactinogenicity and tissue distribution characteristics of Trichinella spiralis lactate dehydrogenase(TsLDH),the TsLDH gene sequence was obtained from GenBank for bioinformatics analysis.Primers were designed to perform RT-PCR amplification,and the PCR products were connected to the pET-30a plasmid to construct the recombinant expression plasmid pET30a-TsLDH,which was then transformed into E.coli BL21(DE3)for expression.After purifying the recombinant protein rTsLDH,reactinogenicity analysis was carried out through Western-blot.Rabbits were immunized by rTsLDH mixed with Freund's adjuvant,and blood was collected to obtain immune serum.The titer of rabbit anti-rTsLDH polyclonal antibody was detected by indirect ELISA and the purified rabbit anti-rTsLDH antibody was used to localize TsLDH.The recombinant protein rTsLDH was successfully expressed under the induction conditions of 37 ℃,200 r/min,and 1 mmol/L IPTG with molecular weight of 39.4 kDa,and it mainly exists in the form of inclusion bodies.Western-blot analysis showed that the recombinant protein rTsLDH can be recognized by Trichinella-infected pig serum with reactinogenicity.The titer of the polyclonal antibody was 1∶5.12×105.The results of tissue localization showed that TsLDH exists in the body surface and rods of Trichinella in the three developmental stages,and it can be secreted around the host intestinal tissue in the adult stage,but not in the infected muscle tissue.The results show that rTsLDH has good reactinogenicity,and TsLDH exists in all stages of the worm,providing a candidate for the diagnostic antigen and drug target screening.关键词
旋毛虫/乳酸脱氢酶/反应原性/免疫组化定位Key words
Trichinella spiralis/lactate dehydrogenase/reactinogenicity/immunohistochemical localization分类
农业科技引用本文复制引用
牛栋玉,张念章,李文卉,刘垠鞠,秦洪涛,张永芝,殷宏,付宝权..旋毛虫乳酸脱氢酶基因的克隆表达及组织定位[J].中国兽医科学,2025,55(4):463-469,7.基金项目
国家重点研发计划项目(2023YFD1801000) （2023YFD1801000）
西藏自治区科技计划项目(QYXTZX-RKZ2024-03-3) （QYXTZX-RKZ2024-03-3）
兰州市青年科技人才创新项目(2023-QN-21) （2023-QN-21）
