本文關(guān)鍵詞：基因優(yōu)化的表達(dá)人輪狀病毒重組腺病毒免疫效果研究　出處：《中國疾病預(yù)防控制中心》2008年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 基因優(yōu)化 輪狀病毒 重組腺病毒 疫苗

【摘要】： A組輪狀病毒(Group A Rotavirus,ARV)是引起全世界嬰幼兒嚴(yán)重腹瀉的最重要病原,在發(fā)展中國家,每年至少約600,000兒童死于輪狀病毒感染。鑒于輪狀病毒危害嚴(yán)重且缺乏有效治療手段,世界衛(wèi)生組織一直將發(fā)展輪狀病毒疫苗列為最優(yōu)先發(fā)展的疫苗項(xiàng)目之一。 由于腺病毒能感染呼吸道和腸道,在誘導(dǎo)全身免疫的同時(shí)產(chǎn)生局部粘膜免疫,安全可靠,可以通過口服或滴鼻給藥,適于嬰幼兒使用,因此,腺病毒載體輪狀病毒基因工程疫苗具有良好的前景。我們實(shí)驗(yàn)室前期利用腺病毒載體表達(dá)輪狀病毒保護(hù)性抗原,對(duì)輪狀病毒基因工程疫苗進(jìn)行了系統(tǒng)研究。我們課題組前期研究表明:用Ad5表達(dá)的輪狀病毒的VP6和VP7基因,采用滴鼻或灌胃的給藥方式,可以在小鼠實(shí)驗(yàn)?zāi)Ｐ蜕先〉昧己玫募?xì)胞免疫和體液免疫效果,并對(duì)輪狀病毒攻擊鼠有一定的保護(hù)作用。但是,由于腺病毒對(duì)輪狀病毒的野生型基因表達(dá)量比較低,因此,增強(qiáng)輪狀病毒抗原的表達(dá),優(yōu)化免疫效果,降低重組腺病毒的用量,提高疫苗的安全性等諸多問題就成了發(fā)展該疫苗的重要課題。 本文通過密碼子優(yōu)化,人工合成了輪狀病毒的VP6,VP7基因,通過對(duì)蛋白表達(dá)量及免疫效果的比較,鑒定了密碼優(yōu)化確實(shí)提高了VP6,VP7蛋白的表達(dá)量,從而減少了病毒的用量。本研究還檢測(cè)了口服腺病毒免疫后,不同時(shí)間點(diǎn)病毒在小鼠體內(nèi)的分布,為該疫苗的進(jìn)一步研究提供了實(shí)驗(yàn)資料。 1.利用密碼子優(yōu)化提高重組腺病毒中人輪狀病毒基因的表達(dá)量 ARV基因的密碼子使用與人類密碼子相差很遠(yuǎn),其AT含量很高。大量研究證明,通過基因密碼改造可以有效提高基因的表達(dá)量。我們對(duì)RV VP6、G1VP7、G2VP7和G3VP7 4個(gè)基因依據(jù)人的偏愛密碼子進(jìn)行了密碼優(yōu)化,人工合成了優(yōu)化基因,利用腺病毒Ad5載體(AdEasy系統(tǒng))在人胚腎細(xì)胞293中進(jìn)行了表達(dá)。結(jié)果顯示,經(jīng)過密碼子優(yōu)化后,與野生型病毒基因相比,4個(gè)基因的蛋白表達(dá)量均有顯著提高。同時(shí),我們對(duì)這4種重組腺病毒進(jìn)行了連續(xù)20代的傳代培養(yǎng),在連續(xù)傳代過程中,插入的輪狀病毒基因一直穩(wěn)定存在和表達(dá)。重組腺病毒rvAdG2VP7(o)在連續(xù)傳代15代后,重組腺病毒rvAdG1VP7(o)和rvAdG2VP7(o)在連續(xù)傳代20代后檢測(cè)到復(fù)制型腺病毒(Replication-Competent AdenovirusRCA)的存在。說明重組腺病毒在293細(xì)胞中連續(xù)傳代具有良好的遺傳穩(wěn)定性,傳代10代以內(nèi)一般檢測(cè)不到RCA,可望滿足腺病毒載體疫苗的研發(fā)需要。 2.密碼子優(yōu)化增強(qiáng)了輪狀病毒VP6基因重組腺病毒的免疫效果 在證實(shí)了通過密碼子優(yōu)化可以提高蛋白表達(dá)量后,我們以VP6基因?yàn)槔?以表達(dá)野生型RVVP6基因的重組病毒rvAdVP6為對(duì)照,在小鼠模型上通過等量病毒(10~8TCID50/只/次)3次滴鼻免疫,觀察了基因優(yōu)化重組腺病毒rvAdVP6(o)的免疫效果。結(jié)果顯示:(1)三次免疫rvAdVP6(o)產(chǎn)生的抗VP6血清IgG抗體水平均明顯高于rvAdVP6,說明優(yōu)化后的重組病毒產(chǎn)生了更強(qiáng)的體液免疫反應(yīng);(2)兩種重組腺病毒均可誘導(dǎo)粘膜免疫,在肺灌洗液、肺勻漿液、腸勻漿液和糞便中均能檢測(cè)出較高水平的特異性IgG和IgA,其中,rvAdVP6(o)肺灌洗液、肺勻漿液和糞便中的IgG和IgA水平均顯著高于rvAdVP6;rvAdVP6(o)腸勻漿液IgG水平顯著高于rvAdVP6,說明優(yōu)化后的病毒產(chǎn)生了更強(qiáng)的粘膜免疫效果;(3)用ELISpot檢測(cè)脾細(xì)胞培養(yǎng)上清中的γ干擾素(IFN-γ),結(jié)果顯示,rvAdVP6(o)產(chǎn)生的斑點(diǎn)數(shù)多于rvAdVP6產(chǎn)生的斑點(diǎn)數(shù),說明優(yōu)化后的病毒產(chǎn)生了更強(qiáng)的細(xì)胞免疫效果;(4)RV攻擊后檢測(cè)小鼠的排毒量,發(fā)現(xiàn)rvAdVP6(o)免疫組的RV排毒減少率明顯高于rvAdVP6,說明優(yōu)化后的病毒對(duì)小鼠的保護(hù)性也增強(qiáng)了。綜上所述,在等量重組腺病毒免疫的情況下,優(yōu)化后的VP6重組病毒在體液免疫、粘膜免疫、細(xì)胞免疫和攻毒保護(hù)方面,均優(yōu)于優(yōu)化前,可望在以后的疫苗應(yīng)用中,達(dá)到減少病毒用量的目的。 3.重組腺病毒口服免疫后在小鼠體內(nèi)的生物分布 為了探討重組腺病毒作為口服疫苗的可行性,研究了經(jīng)灌胃免疫后重組腺病毒在各組織器官中的分布以及抗原表達(dá)情況。將小鼠分為兩組:rvAdVP6(o)免疫組和PBS對(duì)照組,每組70只小鼠,免疫后在7個(gè)時(shí)間點(diǎn)(4h、12h、1d、4d、7d、14d和28d)分別取5只小鼠,采集14種組織標(biāo)本,用免疫組織化學(xué)方法檢測(cè)腺病毒載體和輪狀病毒VP6抗原,用熒光定量PCR檢測(cè)腺病毒載體和輪狀病毒VP6基因的存在。結(jié)果顯示:用重組腺病毒灌胃免疫小鼠后,在4h～28d內(nèi),在肝、腎、脾、心臟、肺、大腸、小腸、胃、食管、舌、腦、氣管、派氏結(jié)和卵巢14種組織標(biāo)本中,均無明顯的病理變化;免疫組織化學(xué)檢測(cè)結(jié)果顯示,只在4h時(shí)在大腸和小腸中檢測(cè)到腺病毒和輪狀病毒VP6抗原;熒光定量PCR在4h時(shí)在大腸、小腸、胃、食管和派氏結(jié)中檢測(cè)到腺病毒載體和輪狀病毒VP6基因;12h時(shí)后在大腸、小腸、胃和食管中仍能檢測(cè)到腺病毒載體和輪狀病毒VP6基因,但其拷貝數(shù)明顯降低;1d后只在大腸和食管中檢測(cè)到少量的腺病毒載體和輪狀病毒VP6基因;在4d、7d后,食管、胃和大腸仍能檢測(cè)到腺病毒載體基因的存在,其他組織中均檢測(cè)不到;至14d時(shí),只有在食管中仍能檢測(cè)到腺病毒載體基因的存在,在其他組織中均檢測(cè)不到。說明灌胃免疫后,腺病毒載體和VP6基因在小鼠體內(nèi)可以表達(dá),并在多種組織器官中存在。 綜上所述,本研究構(gòu)建了4株表達(dá)輪狀病毒密碼子優(yōu)化基因的VP6和VP7的重組腺病毒,與野生型基因相比,其在蛋白表達(dá)水平和免疫效果上均明顯得到提高,在此基礎(chǔ)上檢測(cè)了灌胃免疫后,其在小鼠體內(nèi)的分布情況,這些研究均未見報(bào)道。這些結(jié)果的獲得,為研制我國具有自主知識(shí)產(chǎn)權(quán)的新型輪狀病毒基因工程疫苗進(jìn)一步奠定了基礎(chǔ)。
[Abstract]:Group A rotavirus (Group A, Rotavirus, ARV) is the most important pathogen causing severe diarrhea in infants and young children around the world, in developing countries, at least annually about 600000 children die of rotavirus infection. In view of the serious consequence of rotavirus infection and lack of effective treatment, the WHO has been the development of rotavirus vaccine as one of the highest priority the vaccine project.
Due to adenovirus infection of respiratory and intestinal, local mucosal immunity, in the induction of systemic immunity and is safe and reliable, and can be administered by oral or nasal drops, suitable for use in infants, therefore, adenovirus vector of rotavirus genetic engineering vaccine has a good prospect. Ourprevious using recombinant adenovirus expressing rotavirus protection antigen, gene engineering vaccine against rotavirus were studied. Our preliminary study showed that VP6 and VP7 gene Ad5 expression of rotavirus, by intranasal or intragastric administration of the medication, can achieve good effect of cellular immunity and humoral immunity in mice experimental model, and attack the rotavirus rats have a protective effect. However, the expression of wild type adenovirus on rotavirus gene was relatively low, therefore, enhance the rotavirus antigen expression, immune optimization The effects of reducing the amount of recombinant adenovirus and improving the safety of the vaccine have become an important issue for the development of the vaccine.
The codon optimized synthetic rotavirus VP6 gene, VP7, by comparing the expression and immune effect of protein identification, password optimization can improve the VP6 expression of VP7 protein, thereby reducing the amount of virus. This study also examined oral adenovirus immunity after different time the virus distribution in mice, and provide experimental data for further study on the vaccine.
1. using codon optimization to improve the expression of human rotavirus gene in recombinant adenovirus
ARV gene codon usage and human codon far, its AT content is very high. A large number of studies have shown that the genetic code transformation can effectively improve the expression level of RV. We VP6, G1VP7, G2VP7 and G3VP7 4 gene according to the codon preference of codon optimization, synthetic optimization gene by adenovirus vector Ad5 (AdEasy) in human embryonic kidney cells were expressed in 293. The results showed that after codon optimization, compared with the wild type virus gene, gene expression of 4 proteins were significantly increased. At the same time, we were passaged for 20 generations of these 4 kinds of training recombinant adenovirus, in the process of serial passage, rotavirus gene insertion has been stable. The existence and expression of the recombinant adenovirus rvAdG2VP7 (o) in a row after 15 generations, the recombinant adenovirus rvAdG1VP7 (o) and rvAdG2VP7 (o) detection in continuous after 20 generations The presence of Replication-Competent AdenovirusRCA showed that the recombinant adenovirus had good genetic stability in 293 cell passages. RCA was generally not detected within 10 passages, which is expected to meet the research and development needs of adenovirus vector vaccine.
2. codon optimization enhanced the immune effect of rotavirus VP6 gene recombinant adenovirus
The confirmed by codon optimization can improve protein expression, we used VP6 gene as an example, the recombinant virus rvAdVP6 expression of wild type RVVP6 gene were compared by the same amount virus in mice (10~8TCID50/ / 3) of intranasal immunization were observed and the recombinant adenovirus rvAdVP6 (o the immune effect). The results showed: (1) three rvAdVP6 (o) anti VP6 immune serum antibody level of IgG were significantly higher than that of rvAdVP6, indicating the optimized recombinant virus produced stronger humoral immune response; (2) two kinds of recombinant adenovirus can induce mucosal immunity in lung lavage, lung the homogenate, detected high levels of specific IgG and IgA were intestinal homogenate and feces in the rvAdVP6 (o) in lung lavage fluid and lung homogenate and fecal IgG and IgA levels were significantly higher than that of rvAdVP6; rvAdVP6 (o) IgG intestinal homogenate was significantly higher than that of rvAdVP6, said The optimization of the virus caused a stronger mucosal immune effect; (3) training of interferon gamma in the supernatant of spleen cells by detection of ELISpot (IFN-), rvAdVP6 (o) showed that the spots produced more than the number of number of spots produced by rvAdVP6, shows that the optimized virus produced stronger cellular immune effects; (4) the amount of RV detoxification mice were detected after the attack, rvAdVP6 (o) RV in immune group decreased detoxification rate was significantly higher than rvAdVP6, indicating a protective optimized virus in mice is also enhanced. In summary, in the same amount of recombinant adenovirus immune under the condition of optimized VP6 recombinant virus in humoral immunity. Mucosal immunity, cellular immunity and immune protection, are better than before optimization, the vaccine is expected to in the future application, to reduce the amount of virus.
The biological distribution of 3. recombinant adenovirus in mice after oral immunization
涓轟簡(jiǎn)鎺㈣閲嶇粍鑵虹梾姣掍綔涓哄彛鏈嶇柅鑻楃殑鍙鎬，

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