本文選題：乙型肝炎 + DNA疫苗��； 參考：《中國人民解放軍軍醫(yī)進(jìn)修學(xué)院》2007年博士論文

【摘要】： 慢性乙型肝炎嚴(yán)重危害人類健康，目前尚無特效治療手段。乙肝病毒(HBV)感染人體后發(fā)生慢性化的主要原因是被感染者缺乏有效的特異性細(xì)胞免疫應(yīng)答，不能清除體內(nèi)病毒。由于DNA疫苗在誘導(dǎo)機(jī)體特異性細(xì)胞免疫方面具有獨(dú)特優(yōu)勢，已作為針對慢性乙肝具有潛力的免疫治療手段被廣泛研究。目前乙肝DNA疫苗研究的焦點(diǎn)在于如何誘導(dǎo)足夠強(qiáng)的特異性細(xì)胞免疫，以達(dá)到抑制或消滅病毒、治療乙肝的目的。HBcAg是抗原性最強(qiáng)的HBV結(jié)構(gòu)蛋白，其基因常用于構(gòu)建DNA疫苗，能引發(fā)針對病毒核心蛋白的特異性細(xì)胞免疫反應(yīng)。HBeAg與HBcAg具共線性，是乙肝病毒核心抗原的分泌型。研究表明分泌型HBeAg(P18)誘導(dǎo)免疫耐受，而其胞內(nèi)前體(P20、P22)則有可能誘導(dǎo)較HBcAg更強(qiáng)的細(xì)胞免疫。 本研究課題首先以乙肝病毒前C(Pre-C)-C基因和自身復(fù)制調(diào)控元件增強(qiáng)子Ⅱ(EHNⅡ)為目的基因構(gòu)建疫苗，采用常規(guī)PCR法從adr亞型HBV全基因DNA序列中擴(kuò)增出EHNⅡ-前C-C基因片段，重組到載體VR1012中，構(gòu)建出重組乙肝EHNⅡ/前C-C基因疫苗(VEC)，然后在此DNA疫苗基礎(chǔ)上設(shè)計(jì)使前C基因蛋白產(chǎn)物HBeAg前體151、154、164、167位氨基酸點(diǎn)突變的4對引物，依次加入VEC中，采用單引物二次PCR法進(jìn)行基因點(diǎn)突變，分別得到151點(diǎn)突變(VE1)、151+154點(diǎn)突變(VE2)、151+154+164點(diǎn)突變(VE3)、151+154+164+167點(diǎn)突變(VE4)4種變異體，使前C蛋白水解位點(diǎn)發(fā)生變異，生成前體產(chǎn)物P22、P20后無法被細(xì)胞蛋白酶水解切割產(chǎn)生成熟的分泌型HBeAg。然后將VEC、VE2、VE4分別轉(zhuǎn)染入HepG2細(xì)胞，，ELISA法檢測細(xì)胞裂解液、細(xì)胞培養(yǎng)上清液，結(jié)果均有目的蛋白表達(dá)。細(xì)胞裂解液的HBeAg含量VE4、VE2高于VEC，上清中則反之，VEC細(xì)胞裂解液的HBeAg含量低于上清，VE2、VE4細(xì)胞裂解液的HBeAg含量高于上清；酶免疫細(xì)胞染色定位見目的蛋白主要為胞漿內(nèi)表達(dá)；蛋白免疫印記檢測表達(dá)產(chǎn)物的分子量，結(jié)果表明VEC、VE2、VE4的表達(dá)產(chǎn)物分別為P18、P20、P22左右。分別用VEC、VE2、VE4于0、7、21天肌注方式免疫BALB/C小鼠，同時(shí)設(shè)VEC、VE2、VE4分別聯(lián)合使用插入干擾素γ基因的VR1012質(zhì)粒(VMγ)組 和VEC、VE4各聯(lián)合急性乙肝S基因的DNA疫苗(VAS)免疫動(dòng)物組，ELISA法檢測小鼠抗HBc、抗HBeIgG，發(fā)現(xiàn)免疫接種后第14天即可檢測到抗體，第28天滴度更高，VE2、VE4組抗HBe水平高于VEC組，抗HBc水平無顯著性差異。免疫結(jié)束后(第28天)取小鼠脾細(xì)胞用ELISpot方法和CTL殺傷試驗(yàn)檢測特異性細(xì)胞免疫功能，證實(shí)3種質(zhì)粒均能引發(fā)特異性細(xì)胞免疫反應(yīng)，VE4、VE2強(qiáng)于VEC；聯(lián)用VMγ免疫后能使VEC、VE2、VE4特異性細(xì)胞免疫反應(yīng)增強(qiáng)，聯(lián)用VAS后VE4、VEC特異性細(xì)胞、體液免疫反應(yīng)無增強(qiáng)。本研究結(jié)果為治療型HBVDNA疫苗的進(jìn)一步臨床試驗(yàn)提供了依據(jù)，奠定了基礎(chǔ)。
[Abstract]:Chronic hepatitis B is a serious harm to human health. There is no special therapeutic method at present. The main cause of chronic hepatitis B virus (HBV) infection is the lack of effective specific cellular immune response and the ability to remove the virus in the body. The DNA vaccine has unique advantages in inducing the specific cell immunity of the body. At present, the focus of the study on hepatitis B DNA vaccine is how to induce strong specific cell immunity to inhibit or eliminate the virus..HBcAg is the most antigenic HBV structural protein for the purpose of treating hepatitis B. Its gene is often used to construct DNA vaccine and can be triggered. The specific cellular immune response to virus core protein (.HBeAg) is co linear with HBcAg, which is the secretory type of HBV core antigen. The study shows that secretory HBeAg (P18) induces immune tolerance, and its intracellular precursor (P20, P22) may induce stronger cellular immunity than HBcAg.
First of all, the vaccine was constructed with the pre C (Pre-C) -C gene of hepatitis B virus (HBV) and the self replicating element enhancer II (EHN II) as the target gene, and the EHN II pre C-C gene fragment was amplified from the adr subtype HBV whole gene DNA sequence by the conventional PCR method, and the recombinant hepatitis B EHN II / former gene vaccine was constructed. On the basis of this DNA vaccine, 4 pairs of primers that mutated the 151154164167 amino acid points of the precursor of the precursor of the C gene protein product were designed and added to the VEC, and the single primer two PCR methods were used for gene point mutation, and 151 point mutation (VE1), 151+154 point mutation (VE2), 151+154+164 point mutation (VE3), 151+154+164+167 point mutation (VE4) 4 species were obtained respectively. Variant, the mutation of the hydrolysis site of the former C protein, the production of the precursor product P22, and the production of the mature secretory HBeAg. after P20, and then transfected into HepG2 cells by VEC, VE2 and VE4 respectively, and the cell lysate is detected by ELISA method and the cell culture supernatant has the target protein expression. HBeAg content of the cell lysate is contained. The amount of VE4, VE2 was higher than that of VEC, whereas in the supernatant, the HBeAg content of the VEC cell lysate was lower than that of the supernatant, and the HBeAg content of the VE2, VE4 cell lysate was higher than that of the supernatant; the enzyme immunocell staining localization showed that the target protein was mainly in the cytoplasm, and the protein immuno imprint was used to detect the molecular weight of the expression products, and the results showed that the VEC, VE2, VE4 expression products were respectively. For P18, P20, and P22, VEC, VE2, VE4 were used to immunize BALB/C mice in 0,7,21 days, respectively, and VEC, VE2, and VE4 were combined to insert the interferon gamma gene into the VR1012 plasmids (gamma) group, respectively.
And VEC, VE4 each combined with the DNA vaccine (VAS) of acute hepatitis B S gene vaccine (VAS), the ELISA method was used to detect the anti HBc, anti HBeIgG, and the antibody was detected at fourteenth days after immunization, the titer of the twenty-eighth day was higher, the HBe level of the VE4 group was higher than that of the VEC group, and there was no significant difference. The spleen cells of mice were taken after the end of immunization (twenty-eighth days). The specific cellular immune function was detected by method and CTL killing test. It was confirmed that 3 plasmids could induce specific cellular immune responses, VE4 and VE2 were stronger than VEC, and VM gamma immunization could enhance the immune response of VEC, VE2, VE4 specific cells, combined with VAS VE4, VEC specific cells, and no enhancement in humoral immune response. The results of this study were therapeutic HBVDNA Further clinical trials of the vaccine provided a basis and laid the foundation.

【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【共引文獻(xiàn)】

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1 張敏;辛紹杰;胡燕;侯俊;沈宏輝;王志杰;貌盼勇;;突變型HBV前C-C基因疫苗的構(gòu)建及真核細(xì)胞表達(dá)[J];傳染病信息;2008年05期

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3 郭小兵;;ret基因體外定點(diǎn)突變及其突變效應(yīng)分析[J];醫(yī)藥論壇雜志;2007年12期

4 張寶中;冉多良;張昕;安小平;單云竹;周育森;童貽剛;;用DREAM技術(shù)進(jìn)行全長質(zhì)�？焖俣c(diǎn)突變[J];生物工程學(xué)報(bào);2009年02期

5 張寶中;冉多良;劉大斌;王盛;張昕;安小平;周育森;童貽剛;;利用DREAM設(shè)計(jì)和同源重組進(jìn)行一步定點(diǎn)突變[J];中國生物工程雜志;2008年11期

6 陳波;;一種簡單高效的5’-突出末端平端化方法[J];生物技術(shù);2007年03期

7 吳靜;雷楗勇;張蓮芬;花慧;金堅(jiān);;改善稀有密碼子和氨基酸殘基限制提高重組人ADAM15去整合素結(jié)構(gòu)域蛋白表達(dá)水平[J];微生物學(xué)報(bào);2008年08期

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9 張敏;辛紹杰;段學(xué)章;侯俊;胡燕;沈宏輝;貌盼勇;;單引物二次PCR法對重組質(zhì)粒中HBV前C-C基因進(jìn)行點(diǎn)突變的研究[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2007年04期

10 鄧少麗;程小星;蹇銳;蔣靜;;BCMA基因5'上游序列定點(diǎn)刪除突變對啟動(dòng)子活性的影響[J];中國現(xiàn)代醫(yī)學(xué)雜志;2009年07期

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3 吳靜;重組人ADAM15去整合素結(jié)構(gòu)域蛋白的制備及其作用機(jī)理[D];江南大學(xué);2008年

4 王舉梅;家蠶1型乙酰膽堿酯酶基因（acel）的結(jié)構(gòu)及其功能研究[D];蘇州大學(xué);2013年

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2 管政;CIITA突變體和FasL基因轉(zhuǎn)移抑制MHC-Ⅱ類分子表達(dá)及誘導(dǎo)凋亡的體外研究[D];第二軍醫(yī)大學(xué);2005年

3 郭小兵;RET基因真核表達(dá)載體的構(gòu)建及其體外定點(diǎn)突變[D];鄭州大學(xué);2005年

4 劉U

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