發(fā)布時間：2018-08-16 13:35

【摘要】： 幽門螺桿菌(Helicobacter pylori,Hp)是一種定植于人類胃粘膜的革蘭氏染色陰性、螺桿狀、微需氧菌;在全世界范圍內(nèi)廣泛感染,對人類健康構(gòu)成嚴重危害。Hp的抗感染治療存在較大問題,使用單劑藥物根除率低,而使用多聯(lián)藥物費用昂貴,治愈率不高且不能有效阻止Hp的再感染和復(fù)發(fā),此外,耐藥菌株的增加也使抗Hp治療面臨越來越復(fù)雜的難題,因此研制Hp疫苗和其有效接種可能是預(yù)防Hp感染最有前景的手段。 本研究依托實驗室長期研究Hp疫苗的結(jié)果,擬在已完成的Hp三亞單位融合蛋白HspA-HpaA-UreB414(rHHU)的基礎(chǔ)上,將該基因片段hhu與大腸桿菌不耐熱腸毒素B亞單位基因ltB構(gòu)建融合基因;并通過原核表達系統(tǒng)得到帶分子內(nèi)佐劑LTB的多亞單位蛋白疫苗;純化目的蛋白后對沙鼠進行免疫后攻毒保護實驗,評價該疫苗的免疫保護效率,并與其它疫苗方案的保護效果進行比較,為新型分子內(nèi)佐劑多亞單位Hp疫苗的研制提供一定的實驗依據(jù)。 本實驗完成了以下幾個方面的工作: 1.采用PCR技術(shù)分別擴增目的基因片段hhu和ltB; PCR重疊延伸拼接法將ltB基因融合于hhu基因的頭部,構(gòu)建lhhu融合基因形式;NcoⅠ、XhoⅠ雙酶切后將融合基因構(gòu)建至原核表達載體pET-28a(+)中;經(jīng)酶切、測序鑒定后,陽性重組子分別轉(zhuǎn)化宿主菌E.coli BL21(DE3)、E.coli JM109(DH3)。 2.工程菌分別在25℃、37℃及42℃條件經(jīng)IPTG誘導(dǎo)表達;SDS-PAGE,兔抗LTB、兔抗Hp血清和小鼠抗His_6單抗免疫印跡檢測,證實重組融合蛋白rLHHU在不同溫度條件及不同宿主菌環(huán)境中均表達為兩條分子量接近的蛋白帶,顯示目的蛋白發(fā)生了降解。 3.重新構(gòu)建融合基因hhul形式,將ltB融合于hhu的尾部,并在兩片段間引入8氨基酸柔性linker GGGSGGGS;融合基因構(gòu)建至原核表達載體pET-28a(+)中獲得表達質(zhì)粒phhul;經(jīng)酶切、測序鑒定后,陽性重組子轉(zhuǎn)化宿主菌E.coli BL21(DE3);IPTG誘導(dǎo)工程菌表達,SDS-PAGE及免疫印跡檢測,證實為目的蛋白的表達,分子量約為55.2KD;UVP掃描證實融合蛋白表達約占總蛋白的20%。 4.重組蛋白rHHUL提交PridictProtein(http://cubic.bioc.columbia.edu/predictprotein/)
[Abstract]:Helicobacter pylori (Helicobacter) is a Gram-negative, screw shaped, microaerobic bacterium that is colonized in human gastric mucosa. It is widely infected worldwide and has great problems in anti-infective treatment of H.pylori, which is a serious hazard to human health. The eradication rate of single drug is low, while the cost of multidrug is expensive, the cure rate is not high and can not effectively prevent the reinfection and recurrence of HP. In addition, the increase of drug-resistant strains also makes the treatment of anti-HP more and more complicated. Therefore, the development of HP vaccine and its effective vaccination may be the most promising means to prevent HP infection. Based on the results of a long term study of HP vaccine in laboratory, this study intends to construct the fusion gene based on the completed HP S3 subunit fusion protein HspA-HpaA-UreB414 (rHHU) and the E. coli heat-labile enterotoxin B subunit gene ltB. The multisubunit protein vaccine with intramolecular adjuvant LTB was obtained by prokaryotic expression system. Compared with other vaccine schemes, it provides some experimental basis for the development of novel intramolecular adjuvant multisubunit HP vaccine. This experiment has completed the following aspects of the work: 1. The target gene fragments hhu and ltBwere amplified by PCR, the ltB gene was fused into the head of hhu gene by PCR overlapping extension splicing method, and the lhhu fusion gene form was constructed by double enzyme digestion of NCO 鈪，

本文編號：2186137