摘要
为使大肠杆菌热敏毒素LT的毒素活性丧失的同时仍保留其较强的免疫原性 ,实验依据LT基因的同源序列设计并合成 5条引物 ,采用突出末端PCR法和重组PCR法 ,将克隆于质粒pEWD2 99上的LT基因 ,分别引入BamHI、Ndel和xhol等位点 ,扩增出带有上述RE位点且含有m7和m112 突变点的约 110 0bp和约 80 0bp的DNA片段和不含突变点的约 30 0bp的DNA片段 ,使控制LT毒力活性中心的第 7位和第 112位氨基酸的碱基分别发生诱变 ,各DNA片段经分离、纯化、RE酶切和DNA连接酶连接后 ,插入经BamHI和Xhol双酶切的线性化的高效表达载体pGEX_4T_1的多克隆位点区 ,使LTm基因置于pTac强启动子下且与Thrombin蛋白基因融合表达 ,将连接产物转化入JM10 5菌株 ,挑出可疑阳性菌落 ,提取质粒 ,经酶切鉴定、PCR鉴定和DNA序列分析 ,证明读框正确 ,序列正确 ,获得了pGEX_4T_1(LTm7和pGEX_4T_1(LTm112 两个重组表达质粒。为运用基因工程手段大量生产人和动物大肠杆菌流行性腹泻的疫苗抗原和幽门螺杆菌疫苗的粘膜免疫佐剂 ,完成了基因水平的工作。
In order to loose toxic activity of E.coli heat_labile enterotoxin(LT)and remain its strong immunogenicity,according to the homosequence of LT gene,5 oligonucleotide primers were designed and synthesized in the experiment.Using protuding_end PCR and recombinant PCR,restriction endonu_clease(RE)site of BamH I,NdeI and XhoI were incorporated to LT gene which were cloned to plasmid EWD299,then amplificated 1100 base pair(bp)and 800bp DNA fragments including above RE site and mutational sites of m 7 and m 112 and 300bp DNA fragment unincluding mutational site.Accoring to reproted findings that the 7th and 112th amino acids of LT control toxic activity center of LT,these DNA fragments above were isolated,purificated,cleaved by RE and linked by DNA ligase,then inserted plasmid pGEX_4T_1 cleaved by BamH I and Xhol,this LTm gene and thrombin gene could be high expressed at fusion protein state under the strong promoter Tac.These coustructed plasmids were transfected to E.coli JM105,and positive E.colis were screened.The analysis of DNA Sequencing and RE cleavage showed two recombinant expression plasmid pGEX_4T_1(LTm 7)and pGEX_4T_1(LTm 112 )were constructed rightly and their reading_frames were right.In the experiment,the work of gene level was finished in producing the vaccine antigen of ETEC and the mucosal immunological adjuvant of Helicobacter pylori(Hp)vaccine by genetic engineering.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2002年第6期427-431,共5页
Chinese Journal of Preventive Veterinary Medicine
