基因优化的表达人轮状病毒重组腺病毒免疫效果研究

发布时间：2018-01-01 19:02

  本文关键词：基因优化的表达人轮状病毒重组腺病毒免疫效果研究　出处：《中国疾病预防控制中心》2008年博士论文　论文类型：学位论文

  更多相关文章： 基因优化 轮状病毒 重组腺病毒 疫苗

【摘要】： A组轮状病毒(Group A Rotavirus,ARV)是引起全世界婴幼儿严重腹泻的最重要病原,在发展中国家,每年至少约600,000儿童死于轮状病毒感染。鉴于轮状病毒危害严重且缺乏有效治疗手段,世界卫生组织一直将发展轮状病毒疫苗列为最优先发展的疫苗项目之一。 由于腺病毒能感染呼吸道和肠道,在诱导全身免疫的同时产生局部粘膜免疫,安全可靠,可以通过口服或滴鼻给药,适于婴幼儿使用,因此,腺病毒载体轮状病毒基因工程疫苗具有良好的前景。我们实验室前期利用腺病毒载体表达轮状病毒保护性抗原,对轮状病毒基因工程疫苗进行了系统研究。我们课题组前期研究表明:用Ad5表达的轮状病毒的VP6和VP7基因,采用滴鼻或灌胃的给药方式,可以在小鼠实验模型上取得良好的细胞免疫和体液免疫效果,并对轮状病毒攻击鼠有一定的保护作用。但是,由于腺病毒对轮状病毒的野生型基因表达量比较低,因此,增强轮状病毒抗原的表达,优化免疫效果,降低重组腺病毒的用量,提高疫苗的安全性等诸多问题就成了发展该疫苗的重要课题。 本文通过密码子优化,人工合成了轮状病毒的VP6,VP7基因,通过对蛋白表达量及免疫效果的比较,鉴定了密码优化确实提高了VP6,VP7蛋白的表达量,从而减少了病毒的用量。本研究还检测了口服腺病毒免疫后,不同时间点病毒在小鼠体内的分布,为该疫苗的进一步研究提供了实验资料。 1.利用密码子优化提高重组腺病毒中人轮状病毒基因的表达量 ARV基因的密码子使用与人类密码子相差很远,其AT含量很高。大量研究证明,通过基因密码改造可以有效提高基因的表达量。我们对RV VP6、G1VP7、G2VP7和G3VP7 4个基因依据人的偏爱密码子进行了密码优化,人工合成了优化基因,利用腺病毒Ad5载体(AdEasy系统)在人胚肾细胞293中进行了表达。结果显示,经过密码子优化后,与野生型病毒基因相比,4个基因的蛋白表达量均有显著提高。同时,我们对这4种重组腺病毒进行了连续20代的传代培养,在连续传代过程中,插入的轮状病毒基因一直稳定存在和表达。重组腺病毒rvAdG2VP7(o)在连续传代15代后,重组腺病毒rvAdG1VP7(o)和rvAdG2VP7(o)在连续传代20代后检测到复制型腺病毒(Replication-Competent AdenovirusRCA)的存在。说明重组腺病毒在293细胞中连续传代具有良好的遗传稳定性,传代10代以内一般检测不到RCA,可望满足腺病毒载体疫苗的研发需要。 2.密码子优化增强了轮状病毒VP6基因重组腺病毒的免疫效果 在证实了通过密码子优化可以提高蛋白表达量后,我们以VP6基因为例,以表达野生型RVVP6基因的重组病毒rvAdVP6为对照,在小鼠模型上通过等量病毒(10~8TCID50/只/次)3次滴鼻免疫,观察了基因优化重组腺病毒rvAdVP6(o)的免疫效果。结果显示:(1)三次免疫rvAdVP6(o)产生的抗VP6血清IgG抗体水平均明显高于rvAdVP6,说明优化后的重组病毒产生了更强的体液免疫反应;(2)两种重组腺病毒均可诱导粘膜免疫,在肺灌洗液、肺匀浆液、肠匀浆液和粪便中均能检测出较高水平的特异性IgG和IgA,其中,rvAdVP6(o)肺灌洗液、肺匀浆液和粪便中的IgG和IgA水平均显著高于rvAdVP6;rvAdVP6(o)肠匀浆液IgG水平显著高于rvAdVP6,说明优化后的病毒产生了更强的粘膜免疫效果;(3)用ELISpot检测脾细胞培养上清中的γ干扰素(IFN-γ),结果显示,rvAdVP6(o)产生的斑点数多于rvAdVP6产生的斑点数,说明优化后的病毒产生了更强的细胞免疫效果;(4)RV攻击后检测小鼠的排毒量,发现rvAdVP6(o)免疫组的RV排毒减少率明显高于rvAdVP6,说明优化后的病毒对小鼠的保护性也增强了。综上所述,在等量重组腺病毒免疫的情况下,优化后的VP6重组病毒在体液免疫、粘膜免疫、细胞免疫和攻毒保护方面,均优于优化前,可望在以后的疫苗应用中,达到减少病毒用量的目的。 3.重组腺病毒口服免疫后在小鼠体内的生物分布 为了探讨重组腺病毒作为口服疫苗的可行性,研究了经灌胃免疫后重组腺病毒在各组织器官中的分布以及抗原表达情况。将小鼠分为两组:rvAdVP6(o)免疫组和PBS对照组,每组70只小鼠,免疫后在7个时间点(4h、12h、1d、4d、7d、14d和28d)分别取5只小鼠,采集14种组织标本,用免疫组织化学方法检测腺病毒载体和轮状病毒VP6抗原,用荧光定量PCR检测腺病毒载体和轮状病毒VP6基因的存在。结果显示:用重组腺病毒灌胃免疫小鼠后,在4h～28d内,在肝、肾、脾、心脏、肺、大肠、小肠、胃、食管、舌、脑、气管、派氏结和卵巢14种组织标本中,均无明显的病理变化;免疫组织化学检测结果显示,只在4h时在大肠和小肠中检测到腺病毒和轮状病毒VP6抗原;荧光定量PCR在4h时在大肠、小肠、胃、食管和派氏结中检测到腺病毒载体和轮状病毒VP6基因;12h时后在大肠、小肠、胃和食管中仍能检测到腺病毒载体和轮状病毒VP6基因,但其拷贝数明显降低;1d后只在大肠和食管中检测到少量的腺病毒载体和轮状病毒VP6基因;在4d、7d后,食管、胃和大肠仍能检测到腺病毒载体基因的存在,其他组织中均检测不到;至14d时,只有在食管中仍能检测到腺病毒载体基因的存在,在其他组织中均检测不到。说明灌胃免疫后,腺病毒载体和VP6基因在小鼠体内可以表达,并在多种组织器官中存在。 综上所述,本研究构建了4株表达轮状病毒密码子优化基因的VP6和VP7的重组腺病毒,与野生型基因相比,其在蛋白表达水平和免疫效果上均明显得到提高,在此基础上检测了灌胃免疫后,其在小鼠体内的分布情况,这些研究均未见报道。这些结果的获得,为研制我国具有自主知识产权的新型轮状病毒基因工程疫苗进一步奠定了基础。
[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
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