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首页> 外文期刊>World Journal of Gastroenterology >Construction and characterization of bivalent vaccine candidate expressing HspA and M_r18 000 OMP from Helicobacter pylori
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Construction and characterization of bivalent vaccine candidate expressing HspA and M_r18 000 OMP from Helicobacter pylori

机译：幽门螺杆菌表达HspA和M_r18000 OMP的二价疫苗候选物的构建和表征

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AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with M_r18 000 and heat shock protein A (HspA) from Helicobacter pylori (H. pylori) in E. coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection. METHODS: The target gene of HspA was amplified from H. pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn Ⅰ, BamH Ⅰ simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp_(18), digested by restrictive endonuclease enzyme Hind Ⅲ and BamH Ⅰ simultaneously. pET32a(+)/HspA and Omp_(18) were recovered from 1% agarose gel by gel kit, and ligated with T_4 ligase by BamH Ⅰ digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp_(18) was transformed and expressed in E Coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot. RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3% and 1.4% differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (M_r) of the expressed product was M_r 51 000, M_r of protein expressed by pET32a (+) was about M_r 20 000, and soluble expression product accounted for 18.96% of total bacterial protein. After purification with Ni~(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95%. Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H. pylori and anti-Omp_(18) monocione, suggesting that this protein had good antigenicity. CONCLUSION: The gene coding for H. pylori M_r18 000 OMP and HspA was cloned and expressed successfuly. The results obtained lay the foundation for development of H. pylori protein vaccine and a quick diagnostic kit.
机译：目的：构建能够表达M_r18000的外膜蛋白（OMP）和幽门螺杆菌（H. pylori）的热休克蛋白A（HspA）的重组载体，并探索获得疫苗的可能性。赋予保护免受幽门螺杆菌感染。方法：通过PCR从幽门螺杆菌染色体中扩增HspA的靶基因，并同时通过限制性内切酶kpnⅠ，BamHⅠ将其插入原核表达载体pET32a（+）。用重组载体测序，然后与pET32a（+）/ Omp_（18）一起，分别用限制性核酸内切酶HindⅢ和BamHⅠ消化。通过凝胶试剂盒从1％琼脂糖凝胶中回收pET32a（+）/ HspA和Omp_（18），并通过BamHⅠ消化的粘度末端与T_4连接酶连接。 pET32a（+）/ HspA / Omp_（18）的重组质粒在IPTG的诱导下转化并在大肠杆菌BL21（DE3）中表达。纯化后，通过Western印迹检测融合蛋白的抗原性。结果：酶切分析和测序表明，目的基因已插入到重组载体中，由891个碱基对组成，编码的目标氨基酸为297个氨基酸残基。与Tomb等报道的GenBank相比，幽门螺杆菌的核苷酸序列和氨基酸残基分别有1.3％和1.4％的差异。 SDS-PAGE分析表明，表达产物的相对分子质量（M_r）为M_r 51000，pET32a（+）表达的蛋白质的M_r约为M_r 20000，可溶性表达产物占细菌总蛋白的18.96％。用Ni〜（+2）-NTA琼脂糖树脂纯化后，重组融合蛋白的纯化率为约95％。 Western blot检测表明，重组融合蛋白可以被幽门螺杆菌和抗-Omp_（18）单体的患者血清所识别，表明该蛋白具有良好的抗原性。结论：成功克隆并表达了幽门螺杆菌M_r18000 OMP和HspA基因。获得的结果为开发幽门螺杆菌蛋白疫苗和快速诊断试剂盒奠定了基础。

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