淋球菌LOS 2C7表位筛选及其与HBc融合蛋白的免疫效果评价

发布时间：2018-08-06 17:19

【摘要】： 淋病的病原体是奈瑟菌属(Neisseria)的淋病双球菌(Neisseria gonorrhoeae)。全世界每年有超过6000万新发病例。淋球菌感染可导致一系列疾病,如黏膜炎症,并可能向组织入侵,引起各种组织炎症。淋病同时与艾滋病病毒感染紧密相关。淋病可通过抗生素进行治疗,但随着耐药株的出现,用药物控制淋病面临严峻挑战。疫苗或许是控制淋病最经济、有效的方法,但由于对淋球菌感染过程中免疫反应及重复感染的生物学知之甚少,目前仍没有有效的淋病疫苗面世。因此,有效的靶位筛选是淋球菌疫苗研究的主要方向。本试验将淋球菌的LOS 2C7表位作为主要的疫苗靶位来研究。主要包括以下几方面: (1)淋球菌的LOS 2C7表位的筛选。首先,通过热酚法提取淋球菌的LOS抗原,作为ELISA的包被抗原。其次,本研究将LOS的7个2C7表位进行人工合成,偶联在KLH上,免疫Balb/c雌性小鼠,分别在一免三周、二免后每隔一周进行尾静脉采血,分离血清,进行ELISA检测,评价抗体水平,筛选抗体水平较高的表位。再次,通过人血清介导的补体杀伤试验,评价不同表位的保护力,筛选保护力较好的表位。进一步综合ELISA抗体水平和杀菌试验的结果,确定免疫原性较好的表位。 (2)重组基因与原核表达载体的构建。将确定好的表位基因进行人工合成,表位之间通过linker GSGGSG进行连接,重复3次;通过重叠PCR法对乙肝核心蛋白HBcAg基因的79、80、81位氨基酸基因进行敲除,并将合成的表位基因插入到HBcAg基因MIR区,即上面所述的敲除区。将构建好的HBcAg与表位的重组基因通过原核表达系统进行表达,本试验选择了pET22b载体进行表达,使用BamHⅠ和HindⅢ酶切位点,将构建好的表达载体通过双酶切和菌落PCR进行鉴定,并通过测序进一步确定。 (3)表位与HBcAg融合蛋白的表达与纯化。对构建好的表达载体进行表达条件优化,为方便纯化,促使融合蛋白进行可溶性表达,最终在18℃下进行表达,可得到较好的可溶性表达效果。通过Ni柱进行纯化,BCA法进行蛋白定量,得到免疫动物的融合蛋白抗原。 (4)融合蛋白保护力及免疫效果的评价。纯化后并定量的融合蛋白免疫免疫Balb/c雌性小鼠,分别在一免二周、二免后每隔一周进行尾静脉采血,分离血清,进行ELISA检测,评价融合蛋白的抗体水平。杀菌试验和攻毒试验用于评价融合蛋白的保护力。由于正常Balb/c小鼠对淋球菌不易感,本试验将Balb/c小鼠在攻毒前两天和攻毒后两天分别皮下注射0.5mg17-β雌二醇,从而使Balb/c小鼠对淋球菌易感。进而评价融合蛋白的保护力。综上所述,本试验通过ELISA,杀菌试验,分子生物学技术,攻毒试验,进行了淋球菌LOS表位筛选及其与HBcAg融合蛋白的构建表达,以及保护效果的评价。结果表明筛选的表位与HBcAg融合蛋白能够产生较好的保护力和抗体水平,该试验证明筛选的LOS表位能够为淋球菌的疫苗的研制提供较好的疫苗靶位。
[Abstract]:The pathogen of gonorrhea is the gonorrhea diplococcus (Neisseria gonorrhoeae) of Neisseria. There are more than 6000 million new cases in the world each year. Gonococcal infection can lead to a series of diseases, such as mucous inflammation, and may invade tissue and cause inflammation of various tissues. Gonorrhea is closely related to HIV infection. Gonorrhea can be used. Antibiotics are treated, but with the emergence of drug-resistant strains, drug control of gonorrhea is facing a severe challenge. Vaccine may be the most economical and effective way to control gonorrhea. But there is still no effective gonorrhoeae vaccine due to the lack of biological knowledge about the immune response and repeated infection of gonococcal infection.
Therefore, effective target screening is the main direction of gonococcal vaccine research. In this study, the LOS 2C7 epitope of gonococcus was used as the main vaccine target.
It mainly includes the following aspects:
(1) screening of the LOS 2C7 epitopes of gonococcus. First, the LOS antigen of gonococci was extracted by hot phenol as the antigen of ELISA. Secondly, the 7 2C7 epitopes of LOS were synthesized and coupled to KLH to immunization with Balb/c female mice. The blood was collected in the tail vein for three weeks and every other week after two exempts, and the serum was separated and EL was carried out. ISA was tested to evaluate the level of antibody and screen the epitopes with high level of antibody. Again, the protective ability of different epitopes was evaluated through human serum mediated complement killing test, and the protective ability of the epitopes was screened. The results of ELISA antibody level and bactericidal test were further integrated to determine the better epitopes of immunogenicity.
(2) the construction of the recombinant gene and the prokaryotic expression vector. The epitope was synthesized and the epitopes were connected by linker GSGGSG and repeated for 3 times; the 79,80,81 bit amino acid gene of the hepatitis B core protein HBcAg gene was knocked out by overlapping PCR method and the synthesized epitope gene was inserted into the MIR region of the HBcAg gene, that is, The recombinant gene of the constructed HBcAg and epitopes was expressed through the prokaryotic expression system. The pET22b vector was chosen to express the recombinant gene, and the BamH I and Hind III enzyme cutting sites were used to identify the constructed expression vector by double enzyme cutting and colony PCR, and further confirmed by sequencing.
(3) the expression and purification of the fusion protein of the epitope and HBcAg. The expression condition of the constructed expression vector was optimized to facilitate the purification and the soluble expression of the fusion protein. The expression of the fusion protein was finally expressed at 18 C, and the soluble expression effect was better. The protein was purified by the Ni column and the BCA method was used for the protein quantification to obtain the melting of the immune animals. The antigen of the protein.
(4) evaluation of the protective and immune effects of the fusion protein. The purified and quantified fusion protein immunized Balb/c female mice were immunized in the tail vein for two weeks and after two exempts, respectively. The serum was separated every other week, the ELISA detection was carried out and the antibody level of the fusion protein was evaluated. The bactericidal test and attack test were used to evaluate the fusion protein. Protection. Due to the unsusceptible to gonococcus in normal Balb/c mice, the Balb/c mice were subcutaneously injected with 0.5mg17- beta estradiol for two days before attack and two days after attack, which made the Balb/c mice susceptible to gonococcus, and then evaluated the protective ability of the fusion protein.
To sum up, this experiment was conducted by ELISA, bactericidal test, molecular biology technology and attack test. The LOS epitopes of Neisseria gonorrhoeae and the construction and expression of the fusion protein with HBcAg, as well as the protection effect were evaluated. The results showed that the screened epitopes and HBcAg fusion protein could produce better protection and antibody level. The test proved to be screened. The LOS epitope can provide a better vaccine target for the development of Neisseria gonorrhoeae vaccine.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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