淋球菌LOS 2C7表位筛选及其与HBc融合蛋白的免疫效果评价
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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。
Gonorrhea pathogen is Neisseria gonorrhoeae. There are more than 60 million new cases every year. N.gonorrhoeae infection may lead to a series of diseases, such as mucosal inflammation (urethritis and cervicitis), and the invasion to the organs. Gonorrhea and HIV infection are also closely related. Gonorrhea can be treated with antibiotics, but with the emergence of resistant strains, controlling gonorrhea with drugs faces serious challenges. To control gonorrhea by vaccine maybe the most economical and effective method. But little is known about the immune response during gonococcal infection and the mechanism of repeated infections, there is still no available effective vaccine for gonorrhea.
Therefore, the effective target selection is the main research direction for gonococcal vaccine. N. gonorrhoeae LOS 2C7 epitopes were as major vaccine targets to study in this study, including the following aspects:
(1) Screening N. gonorrhoeae LOS 2C7 epitopes. First, the LOS of N. gonorrhoeae antigen was extracted by hot phenol method, as the ELISA coating antigen. Second, 7 2C7 epitopes of LOS was synthesized and coupled with KLH, and used to immune Balb / c female mice. Sera were separated in first three-week and every week post boosting immunization for ELISA testing and evaluation of antibody level. The higher antibody levels of epitopes were screened. Third, the different protective levels of epitopes were evaluated by human serum complement-mediated killing assay and the higher protective levels of epitopes were screened. The antibody levels were evaluted by ELISA, and the immunogenicity of epitopes was determined by bactericidal tests.
(2) Construction of recombinant gene and prokaryotic expression vector. The three selected epitope genes were synthesized, and the epitopes were connected with the linker GSGGSG, repeated three times. The 79,80,81 amino acids genes of HBcAg were knockout by overlapping PCR method. The epitopes were inserted into the HBcAg gene MIR area. The constructed recombinant HBcAg epitope genes were expressed by prokaryotic expression system. The pET22b expression vector was chosen and BamHⅠand HindⅢrestriction sites were selected. The construction expression vector was identified by restriction enzyme digestion and PCR and sequencing.
(3) Expression and purification of epitope and HBcAg fusion protein. The expression condition was optimized in order to facilitate purification and promote the expression of soluble fusion protein. Soluble expression could be well expressed at 18℃. Fusion protein was purified by Ni column chromatography, and quantitied by BCA method, then used to immunize animals.
(4) The evaluation of the immune effect of fusion proteins. Female Balb/c mice were immunized with purified fusion protein and bleed in first two weeks and each week post boosting immunization. Sera were separated for ELISA testing and evaluation of antibody levels of fusion protein. Bactericidal test and challenge test were used to evaluate the protective effect of fusion protein in Balb/c mice. Because the normal Balb/c mice are not susceptible to gonorrhoeae, the Balb/c mice were subcutaneously treated with 0.5mg17-βestradiol two days before and after challenge, respectively. The protective effect of fusion protein was further evaluated.
In summary, ELISA, bactericidal test, molecular biology techniques, challenge test were carried out in this study for screening N.gonorrhoeae LOS epitopes with HBcAg fusion protein. The results showed that the screened epitopes with HBcAg fusion protein produced a higher antibody levels and better protective effect, and the screened LOS epitopes maybe N. gonorrhoeae vaccine targets.
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。
Gonorrhea pathogen is Neisseria gonorrhoeae. There are more than 60 million new cases every year. N.gonorrhoeae infection may lead to a series of diseases, such as mucosal inflammation (urethritis and cervicitis), and the invasion to the organs. Gonorrhea and HIV infection are also closely related. Gonorrhea can be treated with antibiotics, but with the emergence of resistant strains, controlling gonorrhea with drugs faces serious challenges. To control gonorrhea by vaccine maybe the most economical and effective method. But little is known about the immune response during gonococcal infection and the mechanism of repeated infections, there is still no available effective vaccine for gonorrhea.
Therefore, the effective target selection is the main research direction for gonococcal vaccine. N. gonorrhoeae LOS 2C7 epitopes were as major vaccine targets to study in this study, including the following aspects:
(1) Screening N. gonorrhoeae LOS 2C7 epitopes. First, the LOS of N. gonorrhoeae antigen was extracted by hot phenol method, as the ELISA coating antigen. Second, 7 2C7 epitopes of LOS was synthesized and coupled with KLH, and used to immune Balb / c female mice. Sera were separated in first three-week and every week post boosting immunization for ELISA testing and evaluation of antibody level. The higher antibody levels of epitopes were screened. Third, the different protective levels of epitopes were evaluated by human serum complement-mediated killing assay and the higher protective levels of epitopes were screened. The antibody levels were evaluted by ELISA, and the immunogenicity of epitopes was determined by bactericidal tests.
(2) Construction of recombinant gene and prokaryotic expression vector. The three selected epitope genes were synthesized, and the epitopes were connected with the linker GSGGSG, repeated three times. The 79,80,81 amino acids genes of HBcAg were knockout by overlapping PCR method. The epitopes were inserted into the HBcAg gene MIR area. The constructed recombinant HBcAg epitope genes were expressed by prokaryotic expression system. The pET22b expression vector was chosen and BamHⅠand HindⅢrestriction sites were selected. The construction expression vector was identified by restriction enzyme digestion and PCR and sequencing.
(3) Expression and purification of epitope and HBcAg fusion protein. The expression condition was optimized in order to facilitate purification and promote the expression of soluble fusion protein. Soluble expression could be well expressed at 18℃. Fusion protein was purified by Ni column chromatography, and quantitied by BCA method, then used to immunize animals.
(4) The evaluation of the immune effect of fusion proteins. Female Balb/c mice were immunized with purified fusion protein and bleed in first two weeks and each week post boosting immunization. Sera were separated for ELISA testing and evaluation of antibody levels of fusion protein. Bactericidal test and challenge test were used to evaluate the protective effect of fusion protein in Balb/c mice. Because the normal Balb/c mice are not susceptible to gonorrhoeae, the Balb/c mice were subcutaneously treated with 0.5mg17-βestradiol two days before and after challenge, respectively. The protective effect of fusion protein was further evaluated.
In summary, ELISA, bactericidal test, molecular biology techniques, challenge test were carried out in this study for screening N.gonorrhoeae LOS epitopes with HBcAg fusion protein. The results showed that the screened epitopes with HBcAg fusion protein produced a higher antibody levels and better protective effect, and the screened LOS epitopes maybe N. gonorrhoeae vaccine targets.
引文
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