本文選題：慢性乙型肝炎 + 治療性疫苗��； 參考：《蘭州大學(xué)》2006年碩士論文

【摘要】：慢性乙型肝炎是一種嚴重危害人類健康的全球性疾病,目前尚無有效的藥物進行治療,疫苗仍然是預(yù)防乙肝的重要手段。目前普遍使用的乙型肝炎疫苗是以乙肝表面抗原蛋白為基礎(chǔ)的,此類疫苗可誘導(dǎo)人體產(chǎn)生保護性抗體,從而起到預(yù)防乙型肝炎的作用。但此類疫苗最大的缺點是不能誘導(dǎo)有效的細胞免疫,而細胞免疫在清除細胞內(nèi)病毒,以及打破慢性乙肝患者持續(xù)的免疫耐受狀態(tài)具有極其重要的作用。 本研究對由中國預(yù)防醫(yī)學(xué)科學(xué)院病毒學(xué)研究所構(gòu)建的HBV(中國流行株)核心抗原與PreS1融合重組質(zhì)粒(CS1)進行了轉(zhuǎn)導(dǎo)、培養(yǎng)、誘導(dǎo)表達,并對誘導(dǎo)表達的條件進行了優(yōu)化。通過表達產(chǎn)物的SDS-PAGE電泳、western檢測和抗原性鑒定,確定該蛋白理化、生物學(xué)特性與理論結(jié)果一致。對表達的HBV融合蛋白CS1使用DEAE-Sepharose 4FF離子交換和Sephadex-G75分子篩進行了兩步層析純化得到了純度達到98%以上,抗原性較好,免疫原性較強的蛋白。 將純化后的HBV融合蛋白CS1與蘭州生物制品研究所疫苗三室生產(chǎn)的CHO-乙型肝炎表面抗原(HBsAg)聯(lián)合,以不同劑量免疫BALB/C小鼠,并用兩疫苗單獨免疫作為參照,以生理鹽水作為對照。免疫后于不同時間采血ELISA方法檢測小鼠血清抗體;制備脾臟單個核細胞(MNC),做MTT檢測;用血清抗體效價和淋巴細胞增殖情況兩種標準確定最優(yōu)劑量和最佳免疫程序。用合適劑量的HBcAg與HBsAg聯(lián)合(CS1+S)后,以終濃度為1mg/ml Al(OH)3作為佐劑與不含佐劑的CS1+S抗原組同時免疫BALB/C小鼠,并用兩種疫苗單獨免疫(含和不含Al(OH)3)作為參照,以生理鹽水作為對照。免疫后用ELISA方法測定抗體產(chǎn)生的滴度;制備脾臟單個核細胞(MNC),ELISA法檢測細胞因子IFN-γ分泌水平;應(yīng)用流式細胞儀分析T細胞亞群;最后應(yīng)用DTH試驗檢測抗原引起的BALB/C小鼠遲發(fā)型超敏反應(yīng)。最后的結(jié)果顯示HBcAg與HBsAg聯(lián)合免疫小鼠產(chǎn)生的IFN-γ量與二者單獨免疫相比有顯著性差異(P0.05),CD~(3+)CD~(4+)水平與后者相比顯著增高(P0.05);含Al(OH)_3佐劑的聯(lián)合疫苗CD8~+水平比空白對照組有顯著性差異(P0.05);聯(lián)合疫苗產(chǎn)生的保護性抗體anti-HBs與2倍劑量的HBsAg單獨免疫相比無顯著性差異(P0.05);含Al(OH)_3佐劑與不含佐劑的抗原免疫效果差異不顯著(P0.05)。 總之,本課題工作表明,用CS1與HBsAg聯(lián)合,具有良好的體液免疫與細胞免疫效果,有助于清除HBV病毒并有希望打破HBV感染引起的慢性乙型肝炎病人持續(xù)
[Abstract]:Chronic hepatitis B (CHB) is a global disease that seriously endangers human health. There are no effective drugs to treat chronic hepatitis B. Vaccine is still an important method to prevent hepatitis B. Hepatitis B vaccine, which is widely used at present, is based on hepatitis B surface antigen (HBsAg) protein, which can induce human body to produce protective antibody and thus play a role in the prevention of hepatitis B. But the biggest drawback of this kind of vaccine is that it can not induce effective cellular immunity which plays an extremely important role in clearing the intracellular virus and breaking the persistent immune tolerance in patients with chronic hepatitis B. In this study, the recombinant plasmid of HBV core antigen and PreS1 fusion recombinant plasmid constructed by Institute of Virology, Chinese Academy of Preventive Medical Sciences, was transduced, cultured and expressed, and the conditions of induced expression were optimized. The protein was determined by SDS-PAGE electrophoresis and antigenicity identification. The physicochemical and biological properties of the protein were in agreement with the theoretical results. The expressed HBV fusion protein CS1 was purified by DEAE-Sepharose 4FF ion exchange and Sephadex-G75 molecular sieve by two-step chromatography. The purified HBV fusion protein CS1 was combined with CHO- hepatitis B surface antigen HBsAg produced by Lanzhou Biological products Research Institute. BALB / C mice were immunized with different doses. After immunization, serum antibodies were detected by Elisa method at different time points, MNCX was prepared from spleen mononuclear cells, and MTT assay was performed. The optimal dose and the best immune procedure were determined by two criteria: serum antibody titer and lymphocyte proliferation. BALB / C mice were immunized with appropriate dose of HBcAg and HBsAg combined with CS1 S with final concentration of 1mg/ml Albumin OH3 as adjuvant and CS1 S antigen without adjuvant. The mice were immunized with two kinds of vaccines alone (including and without Albumin OH3) as control and physiological saline as control. After immunization, the titer of antibody was determined by Elisa, the level of cytokine IFN- 緯 was detected by Elisa, the T cell subsets were analyzed by flow cytometry. Finally, DTH test was used to detect delayed type hypersensitivity induced by antigen in BALB / C mice. The results showed that the level of IFN- 緯 produced in mice immunized with HBcAg and HBsAg was significantly higher than that in mice immunized with HBcAg and HBsAg alone. There was no significant difference between the protective antibody anti-HBs produced by the combined vaccine and the 2 times dose of HBsAg alone (P 0.05), and there was no significant difference in the immune effect between the adjuvant containing Albumin and the antigen without the adjuvant (P 0.05). In conclusion, the work of this study shows that the combination of CS1 and HBsAg has good humoral and cellular immune effects, which is helpful to eliminate HBV virus and hopefully break the persistence of chronic hepatitis B patients caused by HBV infection.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392;Q789

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