本文選題：丙型肝炎病毒 + 高變區(qū)1　； 參考：《第二軍醫(yī)大學》2007年碩士論文

【摘要】： 丙型肝炎病毒(hepatitis C virus,HCV)為黃病毒科丙型肝炎病毒屬內(nèi)唯一成員,其基因組是單股正鏈RNA。HCV感染可導致慢性丙型肝炎、肝硬化和肝細胞癌等,對人類健康造成嚴重危害。目前全球約有1.7億HCV感染者,其中我國約有4000萬。聯(lián)合應用干擾素和利巴韋林是目前治療HCV感染的標準方案,但治療費用高,副作用較大,且療效因HCV基因型別的不同而呈現(xiàn)明顯差異。當前HCV疫苗研制尚未取得成功,篩選有效的抗原表位對于HCV疫苗的研發(fā)至關重要。 位于病毒體表面的HCV包膜蛋白2(E2)區(qū)含有中和抗體表位,能誘導產(chǎn)生保護性抗體。高變區(qū)1(hypervariable region 1, HVR1)位于E2的N端,由27個氨基酸殘基組成,序列高度變異, HVR1誘生的抗體無論在體外還是在黑猩猩的實驗中均可以阻斷HCV的感染。曾認為HVR1的變異使得抗體缺乏普遍保護性,然而最近的研究結果顯示,一些不同氨基酸序列的HVR1具有明顯的交叉抗原性,誘生的抗體具有交叉反應性。這為基于HVR1或E2蛋白的HCV疫苗的研制帶來了希望。研究表明,用不同于天然HVR1氨基酸序列的合成肽免疫小鼠所誘導的抗體能與不同株HCV的HVR1發(fā)生交叉反應,這類能模擬來源于不同株HVR1抗原性的表位(模擬表位)在HCV疫苗研究中的應用前景值得探討。 一、HVR1融合蛋白在大腸桿菌中的表達、純化及與HCV陽性血清的反應本研究分別從文獻資料中選取了HVR1的12條模擬表位,將其編碼序列與pET32a原核表達載體中的硫氧還蛋白(Trx)基因融合后插入pET32a載體中,構建的12種表達載體經(jīng)瓊脂糖凝膠電泳、酶切鑒定以及DNA測序證明擴增、拼接序列與預期完全一致。將12種重組表達質(zhì)粒分別轉化大腸桿菌BL21(DE3),用IPTG誘導目的蛋白表達,菌體經(jīng)超聲破碎后進行鎳柱純化,得到了12種HVR1融合蛋白。此外,還表達、純化了空載體pET32a表達的硫氧還蛋白。 收集30份HCV抗體陽性血清,10份正常對照血清,ELISA檢測HVR1融合蛋白與血清的反應。各表位蛋白與10份健康人對照血清反應的OD值均低于0.1。各表位蛋白與30份陽性血清中的大部分能發(fā)生反應,反應率分別為46.7%(P1)、43.3%(P2)、66.7%(P3)、56.7%(P4)、70%(P5)、70%(P6)、53.3%(P7)、56.7%(P8)、70%(P9)、60%(P10)、60%(P11)、53.3%(P12)。這12種表位蛋白聯(lián)合,可與30份血清中的29份發(fā)生陽性反應,反應率升至96.6%。進一步分析發(fā)現(xiàn),P5、P6、P7、P9四種表位蛋白也聯(lián)合可達到上述效果。其中P5、P6、P9均能與30份血清中的21份發(fā)生陽性反應,較其他9種表位蛋白為高。 二、HVR1融合蛋白免疫小鼠及小鼠血清中的抗體檢測。 取上述HVR1融合蛋白以及空載體pET32a表達的硫氧還蛋白分別免疫BALB/c小鼠。融合蛋白與福氏完全佐劑乳化,背部皮下多點注射,200μg/只;2周后,肽與不完全福氏佐劑乳化,以相同劑量免疫,1次/2周,共2次。第8周取小鼠血清進行Western blotting和ELISA檢測。 Western blotting檢測發(fā)現(xiàn),HVR1融合蛋白免疫的BALB/c鼠血清均可與相應的表位蛋白發(fā)生反應,表明血清中存在相應的抗表位蛋白的抗體。 由于ELISA檢測證實P5與丙肝陽性血清的強陽性反應率最高,所以我們合成了P5肽,ELISA方法分析P5肽與12種HVR1融合蛋白免疫BALB/c鼠血清的反應。結果:合成P5肽與其他11種HVR1融合蛋白免疫BALB/c鼠血清可發(fā)生不同程度的交叉反應。與硫氧還蛋白免疫的鼠血清不發(fā)生反應。 ELISA檢測HVR1融合蛋白免疫的BALB/c鼠血清與HVR1合成肽的反應頻率。12種表位蛋白免疫的鼠血清均能與2條具有較大差異J4和H77株的HCV HVR1表位合成肽發(fā)生反應�？蛰d體pET32a表達的硫氧還蛋白免疫BALB/c鼠血清并不與2條HVR1合成肽發(fā)生反應。此結果提示,12種表位蛋白均可誘導出與HCV HVR1表位合成肽具有高交叉頻率的中和抗體。 三、HCV假病毒中和試驗 將H77株pcDNA-HCVE1E2、pCMV-gag-pol、pCMV-GFP轉染HEK293T細胞包裝HCVpp,將含HCVpp的上清感染人肝癌Huh7.5細胞,感染52 h后,于熒光顯微鏡下觀察GFP熒光。 結果顯示,三種質(zhì)粒共轉染HEK293T細胞上清感染的Huh7.5細胞有GFP綠色熒光的表達,表明HEK293T細胞上清中存在感染性的HCV假病毒;含假病毒的上清與P5、P6融合蛋白免疫鼠血清共孵育后感染7.5細胞,則GFP熒光明顯減弱,熒光細胞數(shù)量減少。而含假病毒的上清與Trx免疫鼠血清共孵育后感染Huh7.5細胞,GFP熒光不受影響,表明HVR1融合蛋白免疫的鼠血清能中和HCVpp的感染性。 結論:實驗對12條HCV HVR1模擬表位進行研究,得到的表位蛋白能被多數(shù)HCV患者陽性血清特異識別,接種小鼠后能誘導出針對不同株HVR1的交叉反應性抗體,能中和HCVpp的感染性,提示用HVR1模擬表位可誘導具有交叉反應性的HCV中和抗體,因而在HCV疫苗研制中可能具有潛在的應用價值。
[Abstract]:Hepatitis C virus ( HCV ) is the only member of hepatitis C virus in the family Flaviviridae , and its genome is single strand of positive strand RNA . HCV infection can lead to chronic hepatitis C , liver cirrhosis and hepatocellular carcinoma . There are about 40 million HCV infection in China . There are approximately 170 million HCV infected persons in the world . The combination of interferon and ribavirin is the current standard for the treatment of HCV infection , but the therapeutic cost is high , the side effect is large , and the curative effect is different from the HCV genotype . The development of the current HCV vaccine has not been successful , and the screening of effective epitope is essential for the development of HCV vaccine .






The results showed that HVR1 induced by HVR1 could cross - react with HVR1 of different strains of HCV . The results showed that HVR1 induced by HVR1 could cross - react with HVR1 of different strains of HCV .






One , the expression of HVR1 fusion protein in E . coli , purification and reaction with HCV positive serum were used to select 12 mimic epitope of HVR1 from the literature and then inserted into pET32a vector . The 12 expression vectors were transformed into pET32a vector by agarose gel electrophoresis , enzyme digestion and DNA sequencing . Twelve recombinant expression plasmids were transformed into E . coli BL21 ( DE3 ) .






There were 30 sera of HCV antibody positive serum and 10 normal control serum , and the reaction of HVR1 fusion protein with serum was detected by ELISA . The reaction rates were 46.7 % ( P1 ) , 44.3 % ( P2 ) , 66.7 % ( P6 ) , 55.3 % ( P7 ) , 56.7 % ( P8 ) , 70 % ( P6 ) , 55.3 % ( P7 ) , 65.7 % ( P8 ) , 75.3 % ( P12 ) .






2 . Antibody detection of HVR1 fusion protein in mice and mouse serum .






BALB / c mice were immunized with the above HVR1 fusion protein and the expression of the empty vector pET32a , respectively . The fusion protein was emulsified with Fu ' s complete adjuvant and injected at 200 渭g / 2 for 2 weeks . After 2 weeks , the peptide was immunized with the adjuvant without complete Fu ' s adjuvant at the same dose for 2 times . Western blotting and ELISA were performed on the serum of mice at week 8 .






Western blotting showed that the serum of BALB / c mice immunized with HVR1 fusion protein could react with the corresponding epitope protein , indicating that there was corresponding anti - epitope protein in serum .






The results showed that the serum of BALB / c mice immunized with P - 5 - peptide and eleven HVR1 fusion proteins could cross - react in different degrees .






The serum of BALB / c mice immunized with HVR1 fusion protein and HVR1 synthesized peptide were detected by ELISA . All the 12 epitopes were able to react with two HCV HVR1 epitopes with a larger difference of J4 and H77 . The expression of the empty vector pET32a showed that the serum of BALB / c mice did not react with 2 HVR1 synthetic peptides .






III . neutralization test of HCV pseudovirus






H77 strains pcDNA - HCVE1E2 , pCMV - gag - pol , pCMV - GFP were transfected into HCVPs , and the supernatant was infected with human hepatoma Huh7.5 cells . After 52 h , GFP fluorescence was observed under fluorescence microscope .






The results showed that the expression of GFP green fluorescence in Huh7.5 cells co - transfected with three plasmids showed that infectious HCV pseudoviruses were found in the supernatant , and 7.5 cells were infected with the supernatant of the pseudovirus and the immunized mice with P5 and P6 .






Conclusion : 12 HCV HVR1 epitopes were studied . The results showed that the epitope protein can be specifically identified by the positive serum of most HCV patients . After inoculation of the mice , the cross - reactive antibodies against HVR1 of different strains can be induced , and the HCV neutralizing antibody with cross - reactivity can be induced by HVR1 analog epitope . Therefore , it is possible to have potential application value in the development of HCV vaccine .
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373

【參考文獻】

相關期刊論文 前3條

1 陳青,楊紹基,張曉紅,李剛,姚集魯;用合成肽測定抗丙型肝炎病毒高變區(qū)抗體及其意義[J];廣東醫(yī)學;2002年02期

2 楊劍瑩,金菁,孔玉英,衛(wèi)軍,張祖?zhèn)?李光地,汪垣,袁漢英,李育陽;畢赤酵母表達的重組乙肝表面抗原SS1的純化及性質(zhì)鑒定[J];生物化學與生物物理學報;2000年05期

3 龔育平;高軍;趙平;秦照玲;楊苗;戚中田;;丙型肝炎病毒復合高變區(qū)1模擬表位蛋白的免疫原性分析[J];Virologica Sinica;2006年01期

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本文編號：2048677