捻转血矛线虫苏氨酸醛缩酶结构域包含蛋白的克隆与表达及其体外对山羊外周血单个核细胞功能的影响  

Cloning,expression and effects of Haemonchus contortus aromatic amino acid beta-eliminating lyase threonine aldolase domain containing protein on functions of goat PBMC in vitro

作　　者：温玉玲[1] 王玉俭[1] 吴玲燕[1] 严若峰[1] 徐立新[1] 宋小凯[1] 李祥瑞[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2017年第3期502-508,共7页Journal of Nanjing Agricultural University

基　　金：国家重点基础研究发展计划(973计划)(2015CB150300)

摘　　要：目的]本文旨在探讨捻转血矛线虫苏氨酸醛缩酶结构域包含蛋白(TA-1)体外对山羊外周血单个核细胞(PBMC)功能的影响。[方法]根据TA-1基因序列设计引物进行PCR,将所得目的片段克隆到pMD19-T载体进行测序和分析;将该片段亚克隆到pET-32a(+)载体中构建表达质粒pE T-32a/TA-1。重组子用IPTG诱导后用SDS-PAGE观察其表达情况,对重组蛋白(rT A-1)纯化、连续浓度梯度递减的尿素溶液透析复性。从健康山羊采集的血液分离获得的PBMC与rT A-1(10μg·mL^(-1))共孵育,以rT A-1免疫SD大鼠制备的血清作为一抗,用免疫荧光抗体技术观察rTA-1与山羊PBMC的结合情况;分别用0、10、20和40μg·mL^(-1)的rT A-1刺激PBMC,采用CCK-8法测定细胞增殖情况,采用迁移小室研究细胞迁移情况,用qP CR技术测定各试验组PBMC中IL-2、IL-4、IL-17、IFN-γ及TGF-βmRNA转录情况,采用硝酸还原酶法测定一氧化氮(NO)分泌,采用流式细胞仪检测单核细胞吞噬FITC-dextran的能力。[结果]从捻转血矛线虫总RNA中扩增出约1 194 bp的DNA片段,该基因序列与GenB ank中捻转血矛线虫TA-1基因序列的相似性为95%。SDS-PAGE结果显示该基因成功在大肠杆菌中表达,主要以包涵体形式存在,融合蛋白相对分子质量为64×103,并能与山羊PBMC结合。细胞刺激结果显示rT A-1促进PBMC的增殖(P<0.05)和迁移(P<0.05),刺激细胞因子IL-2、IL-4、IFN-γ和IL-17的表达(P<0.01),增强NO的分泌(P<0.05)。这些影响尤其在rTA-1浓度为20μg·mL^(-1)时极显著(P<0.01),并在40μg·mL^(-1)时极极显著增强单核细胞的吞噬功能(P<0.001)。[结论]rTA-1可作为捻转血矛线虫的一个候选抗原,主要诱导Th2类免疫反应。[ Objectives] This research aims to study the effects of aromatic amino acid beta-eliminating lyase threonine aldolase domain containing protein（ TA-1 ）in Haemonchus contortus on functions of peripheral blood mononuclear ceils （PBMC）from goats in vitro. [ Methods] The gene TA-1 was amplified by PCR using specific primers. Then PCR product was cloned into pMD19-T vector, and the recombinants were identified by PCR and enzyme digestion. The sequence of this gene was compared with that of H. contortus TA-1 available in GenBank. By subcloning the ORF of TA-1 into the vector pET-32a（＋） ,the prokaryotic expression plasmid pET-32a/TA-I was constructed. The recombinants were transformed into E. coli BL21 and induced by IPTG. The expression of recombinant TA-1 （rTA-1 ）was analysed by SDS-PAGE. The rTA-1 was purified with Ni2＋-nitrilotriacetic acid（Ni-NTA） column and refolded by utilizing linear gradient decreased urea. Then purified rTA-1 was administered to SD rats to obtain the anti-rTA-1 sera. Blood samples were collected from healthy goats. PBMC were separated and cultured in vitro with rTA-1 at a final concentration of 10 μg· mL-1. The binding of rTA-1 to PBMC was observed by immunofluorescence antibody technique. After stimulated with rTA-I at a final concentra- tion of 0,10,20 and 40μg.mL-1 ,cell proliferation and migration were tested by CCK-8 method and Millcell~ Hanging Cell Culture Inserts, respectively;the transcriptional levels of IL-2, IL-4,1L-17, IFN-γ and TGF-γ mRNA, NO release of PBMC and monocyte phago- cytosis were examined by real-time quantitative PCR （qPCR）, nitrate reductase assay and flow cytometer, respectively. [ Results ] A gene fragment of 1 194 bp was produced by PCR. It was found that the gene was 95% similar to that of H. contortus TA-1 available in GenBank. SDS-PAGE analysis showed that gene TA-1 was successfully expressed as a fusion protein with a relative molecular weight of64〈 103, mainly in the inclusion body. Immunofluorescence antibody technique ana

关 键 词：捻转血矛线虫 苏氨酸醛缩酶结构域包含蛋白 山羊 外周血单个核细胞 

分 类 号：S852.73[农业科学—基础兽医学]