幽门螺杆菌hp1188基因的原核表达及免疫学鉴定

Prokaryotic expression of Helicobacter pylori gene hp1188 and identification of its immunogenicity

目的:构建高效表达Hp1188重组蛋白的基因工程菌pQE30-hp1188-DH5α,提纯重组蛋白,鉴定其抗原性和免疫原性。通过小鼠灌胃试验,验证Hp1188蛋白阻止幽门螺杆菌(Helicobacter pylori,H.pylori)在胃内定植的作用。方法:构建重组质粒pQE30-hp1188,DNA测序分析正确后,转化大肠杆菌DH5α,IPTG诱导表达。表达蛋白用Ni2+-NTA树脂纯化,SDS-PAGE分析纯度,Bradford法测定浓度,Western blot鉴定对相应抗原的特异性,双向琼脂扩散实验检测效价。纯化重组蛋白和粘膜佐剂霍乱毒素B亚单位(Cholera toxin subunit B,CTB)的混合液灌胃免疫接种预防组BALB/c小鼠,同时设阴性对照组和阳性对照组,免疫4周后用H.pylori NCTC 11637攻击3次,末次攻击4周后处死小鼠,进行胃组织H.pylori培养;H.pylori定植半定量、炎症程度及其炎症活动度的评分,以评价重组蛋白对胃组织H.pylori感染的免疫保护作用。结果:构建的重组表达质粒pQE30-hp1188经DNA测序证实序列完整,插入的基因片段全长810bp,与基因文库中的hp1188基因同源性达98%,并在大肠杆菌DH5α中表达出分子量约为30kD大小的可溶性目的蛋白,表达量占全菌总量的47%,表达蛋白纯化后纯度达90%,浓度为1.0mg/ml。Western blot证实有良好抗原结合特异性,双向琼脂扩散效价为1∶16。预防组H.pylori感染率、定植及炎症程度评分均明显低于阳性对照组(P<0.05)。预防组不仅可以降低H.pylori的定植,而且能减轻H.pylori造成的小鼠胃组织的局部慢性炎症反应。结论:成功构建了基因工程菌pQE30-hp1188-DH5α,并高效表达了Hp1188重组蛋白,表达蛋白具有良好的抗原性和免疫原性,在小鼠体内能阻止H.pylori的定植。

Objective：To construct the gene clones for hp1188 expressed highly in the form of recombinant protein with pQE30 and to determine rHp1188 antigenicity by oral immunization of mice with the recombinant protein. Methods： The hp1188 gene was amplified from helicobacter pylori NCTC 11637 strain by PCR. The PCR product was inserted into prokaryotic expressing plasmid pQE30 and confirmed by DNA sequencing. The recombinant plasmid pQE30-hp1188 was transformed into E. coli DH5α and was induced for expression with IPTG. The recombinant protein was purified by Ni^2＋ -NTA agamsc and analyzed by SDS-PAGE, Bradford quantification, Western blot and double diffusion assays. The mixture of rHp1188 and cholera toxin submlit B was administered orally, designated as the prevention group, while the control groups of mice were designed both for negative and positive, respectively. After 4 weeks of immnization, the positive and prevention groups were. challenged 3 times with H.pylori 11637 and then killed 4 weeks later.The immmloprotection of the gastric mucosa from H. pylori infection was evaluated by assays of H. pylori colonization in vivo and gastritis score. Results： The recombinant expressing vectors pQE30-hpl188 was confirmed by DNA sequencing.The total length of the gene cloned was 810 bp with a 98% sequence homology with that in GeneBank. It was showed by SDS-PAGE that the soluble recombinant fusion protein was about 30 kD in size, representing 47% of total cell protein. Its purity was over 90% after purified with Ni62＋ -NTA agarose with the concentration up to 1.0 mg/ml. Double diffusion test and Western blot analysis confirmed that the antibody titer reached 1 ： 16 with fine specificity against hp1188. The infection rate and colonization of helicobacter pylori in the prevntion group was much lower than that of the positive at the same period of infection（ P 〈 0.05） .Reliefs of the local chronic inflammation in gastric tissues of the mice were also observed in the prevention group. Conclusion： The recombinant expressing vectors pQE30-hp1188-DH5α are successfully constructed. The recombinant Hp1188 protein is highly expressed. The recombinant protein possesses fine antigenicity, and immunization with the recombinant protein is able to inhibit adhesive capacity of H.pylori in vivo.