淋球菌LOS 2C7表位篩選及其與HBc融合蛋白的免疫效果評價(jià)
發(fā)布時(shí)間：2018-08-06 17:19
【摘要】： 淋病的病原體是奈瑟菌屬(Neisseria)的淋病雙球菌(Neisseria gonorrhoeae)。全世界每年有超過6000萬新發(fā)病例。淋球菌感染可導(dǎo)致一系列疾病,如黏膜炎癥,并可能向組織入侵,引起各種組織炎癥。淋病同時(shí)與艾滋病病毒感染緊密相關(guān)。淋病可通過抗生素進(jìn)行治療,但隨著耐藥株的出現(xiàn),用藥物控制淋病面臨嚴(yán)峻挑戰(zhàn)。疫苗或許是控制淋病最經(jīng)濟(jì)、有效的方法,但由于對淋球菌感染過程中免疫反應(yīng)及重復(fù)感染的生物學(xué)知之甚少,目前仍沒有有效的淋病疫苗面世。 因此,有效的靶位篩選是淋球菌疫苗研究的主要方向。本試驗(yàn)將淋球菌的LOS 2C7表位作為主要的疫苗靶位來研究。 主要包括以下幾方面: (1)淋球菌的LOS 2C7表位的篩選。首先,通過熱酚法提取淋球菌的LOS抗原,作為ELISA的包被抗原。其次,本研究將LOS的7個(gè)2C7表位進(jìn)行人工合成,偶聯(lián)在KLH上,免疫Balb/c雌性小鼠,分別在一免三周、二免后每隔一周進(jìn)行尾靜脈采血,分離血清,進(jìn)行ELISA檢測,評價(jià)抗體水平,篩選抗體水平較高的表位。再次,通過人血清介導(dǎo)的補(bǔ)體殺傷試驗(yàn),評價(jià)不同表位的保護(hù)力,篩選保護(hù)力較好的表位。進(jìn)一步綜合ELISA抗體水平和殺菌試驗(yàn)的結(jié)果,確定免疫原性較好的表位。 (2)重組基因與原核表達(dá)載體的構(gòu)建。將確定好的表位基因進(jìn)行人工合成,表位之間通過linker GSGGSG進(jìn)行連接,重復(fù)3次;通過重疊PCR法對乙肝核心蛋白HBcAg基因的79、80、81位氨基酸基因進(jìn)行敲除,并將合成的表位基因插入到HBcAg基因MIR區(qū),即上面所述的敲除區(qū)。將構(gòu)建好的HBcAg與表位的重組基因通過原核表達(dá)系統(tǒng)進(jìn)行表達(dá),本試驗(yàn)選擇了pET22b載體進(jìn)行表達(dá),使用BamHⅠ和HindⅢ酶切位點(diǎn),將構(gòu)建好的表達(dá)載體通過雙酶切和菌落PCR進(jìn)行鑒定,并通過測序進(jìn)一步確定。 (3)表位與HBcAg融合蛋白的表達(dá)與純化。對構(gòu)建好的表達(dá)載體進(jìn)行表達(dá)條件優(yōu)化,為方便純化,促使融合蛋白進(jìn)行可溶性表達(dá),最終在18℃下進(jìn)行表達(dá),可得到較好的可溶性表達(dá)效果。通過Ni柱進(jìn)行純化,BCA法進(jìn)行蛋白定量,得到免疫動物的融合蛋白抗原。 (4)融合蛋白保護(hù)力及免疫效果的評價(jià)。純化后并定量的融合蛋白免疫免疫Balb/c雌性小鼠,分別在一免二周、二免后每隔一周進(jìn)行尾靜脈采血,分離血清,進(jìn)行ELISA檢測,評價(jià)融合蛋白的抗體水平。殺菌試驗(yàn)和攻毒試驗(yàn)用于評價(jià)融合蛋白的保護(hù)力。由于正常Balb/c小鼠對淋球菌不易感,本試驗(yàn)將Balb/c小鼠在攻毒前兩天和攻毒后兩天分別皮下注射0.5mg17-β雌二醇,從而使Balb/c小鼠對淋球菌易感。進(jìn)而評價(jià)融合蛋白的保護(hù)力。 綜上所述,本試驗(yàn)通過ELISA,殺菌試驗(yàn),分子生物學(xué)技術(shù),攻毒試驗(yàn),進(jìn)行了淋球菌LOS表位篩選及其與HBcAg融合蛋白的構(gòu)建表達(dá),以及保護(hù)效果的評價(jià)。結(jié)果表明篩選的表位與HBcAg融合蛋白能夠產(chǎn)生較好的保護(hù)力和抗體水平,該試驗(yàn)證明篩選的LOS表位能夠?yàn)榱芮蚓囊呙绲难兄铺峁┹^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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：2168423
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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，本文編號：2168423
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