本文選題：乙型肝炎 + 熱休克蛋白　； 參考：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2011年博士論文

【摘要】：慢性乙型肝炎(CHB)嚴(yán)重危害人類健康,臨床上尚無(wú)理想治療手段,目前主要用核苷類藥物和干擾素進(jìn)行治療,但是并不能完全治愈乙型肝炎,且長(zhǎng)期使用會(huì)使HBV產(chǎn)生耐藥突變并具有較大的副作用。乙型肝炎病毒感染人體后發(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,無(wú)法清除體內(nèi)的病毒。DNA疫苗由于在誘導(dǎo)機(jī)體特異性細(xì)胞免疫應(yīng)答方面的優(yōu)勢(shì),被廣泛關(guān)注。但是經(jīng)過(guò)多年來(lái)的研究,DNA疫苗仍未走進(jìn)臨床,重要原因之一就是隨著受試動(dòng)物體形的增大,DNA疫苗誘導(dǎo)免疫應(yīng)答的能力迅速下降；此外缺乏滿意的遞送方式也是DNA疫苗發(fā)展的主要阻礙。因此,如何在安全的前提下,采取有效的遞送方式和加強(qiáng)誘導(dǎo)的免疫應(yīng)答,特別是加強(qiáng)細(xì)胞免疫應(yīng)答水平,是目前研制有效DNA疫苗的關(guān)鍵問(wèn)題。 使用分枝桿菌熱休克蛋白70羧基端(HSP70c)或T細(xì)胞刺激表位(HSP70t)基因構(gòu)建融合基因疫苗可以起到佐劑的作用,同時(shí)減少機(jī)體針對(duì)HSP70的免疫應(yīng)答。利用攜帶DNA質(zhì)粒的減毒傷寒沙門菌口服,可直接將DNA質(zhì)粒遞送至抗原呈提細(xì)胞,極大提高DNA疫苗的利用率,同時(shí)口服的免疫方式安全方便,易于為患者接受。使用DNA疫苗初次免疫-痘苗病毒Ankara株(MVA)載體疫苗加強(qiáng)免疫的策略也可以有效增強(qiáng)機(jī)體的免疫應(yīng)答。 采取多種免疫增強(qiáng)策略綜合應(yīng)用,可以更好的優(yōu)化DNA疫苗的免疫效果。本研究通過(guò)HBV基因與HSP70c或HSP70t基因構(gòu)建融合基因疫苗、使用口服減毒傷寒沙門菌遞送DNA疫苗和利用MVA載體疫苗強(qiáng)化免疫等多種手段,探索提高HBV DNA疫苗誘導(dǎo)特異性免疫應(yīng)答的更佳優(yōu)化條件。 首先,將乙肝病毒preC/C基因或preS2/S基因、HSP70c基因或HSP70t基因分別先后插入VR1012載體,獲得含HBV preC/C-HSP70c、HBV preC/C-HSP70t、HBV preS2/S-HSP70c和HBV preS2/S-HSP70t融合基因的DNA疫苗。再將構(gòu)建好的重組DNA疫苗轉(zhuǎn)染HepG2細(xì)胞,ELISA法和化學(xué)發(fā)光法檢測(cè)目的抗原,證實(shí)重組DNA疫苗可以正確表達(dá)目的蛋白并具有抗原性。然后,將重組DNA疫苗電轉(zhuǎn)化減毒傷寒沙門菌SL7207,獲得穩(wěn)定的攜帶重組DNA疫苗的重組減毒沙門菌。最后,分別采用HBV DNA疫苗肌肉注射初次免疫+MVA載體疫苗腹腔注射加強(qiáng)免疫、HBV-HSP70融合DNA疫苗肌肉注射初次免疫+MVA載體疫苗腹腔注射加強(qiáng)免疫、攜帶HBV DNA疫苗的重組減毒沙門菌口服初次免疫+MVA載體疫苗腹腔注射加強(qiáng)免疫和攜帶HBV-HSP70融合DNA疫苗的重組減毒沙門菌口服初次免疫+MVA載體疫苗腹腔注射加強(qiáng)免疫的方案,免疫Balb/c小鼠,收集血清和脾淋巴細(xì)胞檢測(cè)特異性免疫應(yīng)答。 體液免疫方面,采用ELISA檢測(cè)血清特異性抗體,結(jié)果顯示各實(shí)驗(yàn)組均可誘導(dǎo)小鼠產(chǎn)生特異性抗體,抗體產(chǎn)生的時(shí)間和水平的差異無(wú)統(tǒng)計(jì)學(xué)意義。細(xì)胞免疫方面通過(guò)流式細(xì)胞術(shù)檢測(cè)CD3+CD4+/CD3+CD8+T細(xì)胞比值評(píng)估非特異性細(xì)胞免疫水平,ELISpot檢測(cè)肽刺激脾淋巴細(xì)胞分泌IFN-γ評(píng)估特異性細(xì)胞免疫效果,結(jié)果顯示無(wú)論是特異性還是非特異性細(xì)胞免疫,HBV-HSP70融合DNA疫苗優(yōu)于普通DNA疫苗,HBV-HSP70t融合DNA疫苗優(yōu)于HBV-HSP70c融合DNA疫苗。使用減毒沙門菌口服遞送DNA疫苗與裸DNA疫苗肌肉注射所誘導(dǎo)的細(xì)胞免疫應(yīng)答水平相當(dāng)。實(shí)驗(yàn)結(jié)果為治療性HBV DNA疫苗的應(yīng)用研究打下了基礎(chǔ)。
[Abstract]:Chronic hepatitis B is a serious harm to human health . There is no ideal treatment in clinic , but it can not cure hepatitis B completely , but it can ' t cure hepatitis B completely . The main reason for chronic infection after hepatitis B virus infection is the lack of effective specific cellular immunity . The DNA vaccine has not yet entered the clinic because of its superiority in inducing body specific cellular immune response . One of the important reasons is that the ability of DNA vaccine to induce immune response is rapidly decreased with the increase of the body shape of the animal .
In addition , the lack of satisfactory delivery methods is the main obstacle to the development of DNA vaccine . Therefore , it is a key problem to develop effective DNA vaccine under the precondition of safety , effective delivery methods and enhanced immune response , especially the level of cellular immune response .


By taking the attenuated Salmonella typhimurium carrying the DNA plasmid to take orally , the DNA plasmid can be directly delivered to the antigen presenting cell , so that the utilization rate of the DNA vaccine can be greatly improved , the oral immunization mode is safe and convenient , and the immune response of the organism can be effectively enhanced .


In this study , a fusion gene vaccine was constructed by combining HBV gene and HSP70t gene , DNA vaccine was delivered by oral attenuated Salmonella typhimurium , and immunity was enhanced by using MVA vector vaccine . The optimal conditions for improving the specific immune response of HBV DNA vaccine were explored .


The recombinant attenuated Salmonella typhimurium SL7207 containing HBV preC / C - HSP70t , HBV preS2 / S - HSP70t , HBV preS2 / S - HSP7070 and HBV preS2 / S - HSP70t fusion gene was obtained .


In the aspect of humoral immunity , the serum - specific antibody was detected by ELISA . The results showed that all experimental groups could induce specific antibody and antibody production in mice . The results showed that the specific cell immunity was assessed by flow cytometry . The results showed that HBV - HSP70 fusion DNA vaccine was superior to common DNA vaccine . The results showed that the DNA vaccine was superior to HBV - HSP70t fusion DNA vaccine .

【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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