基于HCMV-gB抗原的疫苗分子設(shè)計及其免疫效果評價
發(fā)布時間：2019-02-19 20:38
【摘要】：人巨細胞病毒(Human cytomegalovirus,HCMV)屬于皰疹病毒科β亞科病毒,其感染對胎兒和免疫功能低下者危害嚴(yán)重。接種疫苗可能是某些高危人群預(yù)防和控制HCMV感染的有效措施�；诓《颈砻嫣堑鞍譯B的基因工程疫苗在孕期婦女與移植患者的二期臨床試驗中顯示了良好的保護效果。為了進一步增強gB疫苗的免疫保護效果,本研究對新型gB候選疫苗抗原進行了分子設(shè)計和免疫效果評價。 根據(jù)已經(jīng)發(fā)表的HSV-1-gB的晶體結(jié)構(gòu),通過SWISS-Prot蛋白質(zhì)晶體數(shù)據(jù)庫在線模擬得到了gB的三維結(jié)構(gòu)。模擬結(jié)果表明,已經(jīng)發(fā)現(xiàn)的所有三個中和表位區(qū)AD-1、AD-2和DLD都位于gB分子的膜遠端球狀結(jié)構(gòu)域。根據(jù)三維模擬結(jié)構(gòu)和維持球狀結(jié)構(gòu)域構(gòu)象的原則,本研究設(shè)計了gB1和gB2兩個新型候選疫苗抗原。通過重組PCR擴增出編碼gB1和gB2的融合基因片段,將其定向克隆到哺乳動物細胞表達載體pCI-neo中,用貼壁培養(yǎng)的293FT細胞進行蛋白的瞬時表達,并通過鎳柱親和層析純化目的蛋白。免疫印跡試驗表明純化的兩個重組目的蛋白具有抗原性。 以CpG-ODN + Al(OH)3作為gB1和gB2的免疫佐劑,通過兩針肌肉注射免疫BALB/c小鼠,在五周內(nèi)快速制備獲得了兩個重組蛋白的高效價免疫血清,經(jīng)ELISA檢測抗原特異IgG抗體滴度分別為1:6400～1:51200和1:25600～1:51200。用免疫血清對表達GFP的HCMV-VR1578病毒進行微量中和試驗,結(jié)果表明:無補體存在時gB1和gB2免疫血清50%中和滴度分別為1:51.8和1:24.1,加入家兔補體后分別為1:744和1:264。 綜上所述,本研究成功地設(shè)計并驗證了兩個具有良好應(yīng)用前景的新型HCMV-gB候選疫苗抗原,為進一步研究HCMV亞單位疫苗奠定了基礎(chǔ)。
[Abstract]:Human cytomegalovirus (Human cytomegalovirus,HCMV) belongs to 尾 subfamily of herpesvirus family. Vaccination may be an effective measure to prevent and control HCMV infection in some high-risk population. The genetic engineering vaccine based on virus surface glycoprotein (gB) has shown good protective effect in the second phase clinical trial of pregnant women and transplant patients. In order to further enhance the immune protection of gB vaccine, molecular design and immunological evaluation of novel gB vaccine antigen were carried out in this study. According to the published crystal structure of HSV-1-gB, the 3D structure of gB was obtained by on-line simulation of SWISS-Prot protein crystal database. The simulation results show that all three neutralizing epitopes, AD-1,AD-2 and DLD, are located in the spherical domain at the distal end of the membrane of the gB molecule. According to the principle of three-dimensional simulation and maintaining conformation of spherical domain, two novel candidate vaccine antigens, gB1 and gB2, were designed in this study. The fusion gene encoding gB1 and gB2 was amplified by recombinant PCR and cloned into mammalian cell expression vector pCI-neo. Transient expression of the protein was carried out by adherent 293FT cells. The target protein was purified by nickel affinity chromatography. Western blot analysis showed that the two recombinant proteins were antigenicity. Using CpG-ODN Al (OH) 3 as the adjuvant of gB1 and gB2, the high titer immune serum of two recombinant proteins was prepared by intramuscular injection of BALB/c mice in five weeks. The titers of antigen-specific IgG antibody detected by ELISA were 1: 6400, 1: 51200 and 1: 25600, 1: 51 200, respectively. The neutralization test of HCMV-VR1578 virus expressing GFP with immune serum was carried out. The results showed that the neutralization titers of gB1 and gB2 immunized serum were 1: 51.8 and 1: 24.1, respectively, in the absence of complement, the neutralization titers of gB1 and gB2 were 1: 51.8 and 1: 24.1, respectively. After adding rabbit complement, it was 1: 744 and 1: 264, respectively. In conclusion, two novel HCMV-gB candidate vaccine antigens were successfully designed and validated, which laid a foundation for further study of HCMV subunit 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】：2011
【分類號】：R392
本文編號：2426855
[Abstract]:Human cytomegalovirus (Human cytomegalovirus,HCMV) belongs to 尾 subfamily of herpesvirus family. Vaccination may be an effective measure to prevent and control HCMV infection in some high-risk population. The genetic engineering vaccine based on virus surface glycoprotein (gB) has shown good protective effect in the second phase clinical trial of pregnant women and transplant patients. In order to further enhance the immune protection of gB vaccine, molecular design and immunological evaluation of novel gB vaccine antigen were carried out in this study. According to the published crystal structure of HSV-1-gB, the 3D structure of gB was obtained by on-line simulation of SWISS-Prot protein crystal database. The simulation results show that all three neutralizing epitopes, AD-1,AD-2 and DLD, are located in the spherical domain at the distal end of the membrane of the gB molecule. According to the principle of three-dimensional simulation and maintaining conformation of spherical domain, two novel candidate vaccine antigens, gB1 and gB2, were designed in this study. The fusion gene encoding gB1 and gB2 was amplified by recombinant PCR and cloned into mammalian cell expression vector pCI-neo. Transient expression of the protein was carried out by adherent 293FT cells. The target protein was purified by nickel affinity chromatography. Western blot analysis showed that the two recombinant proteins were antigenicity. Using CpG-ODN Al (OH) 3 as the adjuvant of gB1 and gB2, the high titer immune serum of two recombinant proteins was prepared by intramuscular injection of BALB/c mice in five weeks. The titers of antigen-specific IgG antibody detected by ELISA were 1: 6400, 1: 51200 and 1: 25600, 1: 51 200, respectively. The neutralization test of HCMV-VR1578 virus expressing GFP with immune serum was carried out. The results showed that the neutralization titers of gB1 and gB2 immunized serum were 1: 51.8 and 1: 24.1, respectively, in the absence of complement, the neutralization titers of gB1 and gB2 were 1: 51.8 and 1: 24.1, respectively. After adding rabbit complement, it was 1: 744 and 1: 264, respectively. In conclusion, two novel HCMV-gB candidate vaccine antigens were successfully designed and validated, which laid a foundation for further study of HCMV subunit 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】：2011
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前1條
1 羅丹;方峰;;人巨細胞病毒疫苗的相關(guān)研究進展[J];傳染病信息;2006年03期
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