仿刺参补体AjC3部分基因的原核表达及多克隆抗体的制备  

Prokaryotic Expression of Gene Fragments of AjC3 and Preparation of Polyclonal Antibody in Sea Cucumber Apostichopus japonicus

作　　者：张思[1] 李明[1] 张建清[1] 卢亚楠[1] 翟钰[1] 张峰[1] ZHANG Si LIMing ZHANG Jianqing LU Yanan ZHAI Yu ZHNAG Feng(Fisheries and Life College, Dalian Ocean University, Dalian 116023, China)

机构地区：[1]大连海洋大学水产与生命学院,辽宁大连116023

出　　处：《水产科学》2017年第1期94-98,共5页Fisheries Science

基　　金：国家自然科学基金资助项目(30471223)

摘　　要：通过制备仿刺参补体C3(AjC3)多克隆抗体,为进一步研究仿刺参补体AjC3免疫机制奠定基础。利用PCR技术扩增AjC3部分基因片段(4556~5110bp),将该片段与原核表达载体pGS-21a连接。将重组表达质粒转化到Transetta(DE3)中经IPTG诱导表达。表达的重组蛋白经镍柱纯化后,作为抗原免疫小鼠制备AjC3多克隆抗体。分别用间接ELISA,Western blot检测抗体的效价和特异性。结果显示,PCR扩增得到约555bp的目的片段,重组蛋白分子量大小约56ku;间接ELISA检测抗体效价达1∶25600,Western blot结果显示,多克隆抗体具有良好的特异性。该试验成功的制备了补体AjC3多克隆抗体,为补体AjC3的进一步研究提供了检测工具。The polyclonal antibody of complement component C3 （AjC3） was prepared to provide references for further research on immunologic mechanism in sea cucumber Apostichopus japonicus. The gene frag- ments of AjC3 was amplified by PCR and inserted into the prokaryotic expression vector pGS-21a. The re- combinant plasmid was transformed into the transetta（DE3） and induced to express by IPTG. The expres- sion products were purified by Ni-NTA. The recombinant protein as antigen was employed to immunize mice to prepare the polyclonal antibody. The title and specificity of the polyclonal antibody were deter- mined by indirect ELISA and Western blot. The results showed that the length of PCR product was 555 bp. The size of recombinant protein was 56 ku. The title was 1 ：25600 by indirect ELISA. Western blot proved that the polyclonal antibody had good specificity and was successfull prepared, which provided helpful tools for further researches on AjC3.

关 键 词：仿刺参 AjC3 多克隆抗体 原核表达 

分 类 号：S968.9[农业科学—水产养殖]