發(fā)布時(shí)間：2018-01-06 15:23

  本文關(guān)鍵詞：輪狀病毒抗原蛋白VP4基因重組載體疫苗研究　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 輪狀病毒抗原基因VP4 重疊PCR 穿梭載體pBEX/VP4 腸袢實(shí)驗(yàn) 口服疫苗

【摘要】： 輪狀病毒(rotavirus,RV)感染是引起嬰幼兒重癥腹瀉最重要的因素之一,也是造成發(fā)展中國家兒童死亡的重要原因。在世界各國,90%以上的嬰兒在3歲之前均受到了RV的感染,在5歲以下腹瀉住院患兒中,RV感染病例占20%--70%,5歲以上的兒童幾乎全都感染過RV。美國每年與輪狀病毒感染有關(guān)的醫(yī)療費(fèi)用高達(dá)3—10億元,可見輪狀病毒腹瀉不僅影響了全球兒童的健康,而且造成嚴(yán)重的經(jīng)濟(jì)負(fù)擔(dān)。有效的疫苗接種可以顯著減少由RV引起的兒童重癥腹瀉,也是唯一可行的預(yù)防控制RV較高發(fā)病率和死亡率的方法[1-2]。 輪狀病毒顆粒由3層組成:外部衣殼、內(nèi)部衣殼和核心。外部衣殼由VP7和VP4組成。VP7形成相對(duì)光滑的外表面,VP4組成釘狀突起,二者都是中和抗原,可誘導(dǎo)機(jī)體產(chǎn)生中和抗體。VP4還在病毒感染過程中扮演重要的作用,作為一種細(xì)胞結(jié)合蛋白,VP4經(jīng)胰酶切割后可增強(qiáng)輪狀病毒對(duì)宿主的感染性。因此,以輪狀病毒外殼蛋白基因VP4基因,將其重組到原核表達(dá)載體上,以研究病原基因在宿主菌中的表達(dá)情況,為新型口服疫苗的研究奠定基礎(chǔ)[3-5]。 目的:構(gòu)建攜帶輪狀病毒外殼蛋白VP4基因的重組質(zhì)粒(即pBEX/VP4),檢測重組質(zhì)粒的表達(dá),并對(duì)重組載體表達(dá)蛋白的安全性進(jìn)行驗(yàn)證。 方法:(1)根據(jù)Genbank中鼠源VP4的基因序列設(shè)計(jì)34對(duì)引物,通過重疊PCR合成VP4基因。以pGEX-5x-1為基礎(chǔ),構(gòu)建穿梭表達(dá)載體pBEX,并將合成的VP4基因重組入pBEX,得到重組質(zhì)粒pBEX/VP4。(2)將其轉(zhuǎn)化大腸桿菌,SDS-PAGE驗(yàn)證蛋白的表達(dá)。通過腸袢實(shí)驗(yàn)驗(yàn)證表達(dá)蛋白的安全性。 結(jié)果:(1)測序表明,合成的VP4基因正確,并成功構(gòu)建了穿梭載體pBEX/VP4。(2)VP4蛋白在大腸桿菌中成功表達(dá),其表達(dá)蛋白經(jīng)腸袢實(shí)驗(yàn)證實(shí)是無毒的。 結(jié)論:(1)在沒有模板的情況下,重疊PCR法可以用來合成目的基因;(2)VP4蛋白在大腸桿菌中的表達(dá),說明穿梭載體的構(gòu)建是成功的,而腸袢實(shí)驗(yàn)驗(yàn)證了表達(dá)蛋白的安全性,為以后將重組穿梭載體轉(zhuǎn)入宿主菌,作為新型口服疫苗的研究奠定了基礎(chǔ)。
[Abstract]:Rotavirus (rotavirus, RV) infection is one of the most important factors of severe diarrhea in infants, but also an important cause of death in children in developing countries. In the world, more than 90% babies have been infected with RV before the age of 3, in the following 5 years old hospitalized children with diarrhea, RV infections accounted for 20%--70% 5 children over the age of almost all RV. infected each year in the United States with rotavirus infection related medical expenses up to 3 to 1 billion yuan, visible rotavirus diarrhea not only affects the global children's health, but also caused serious economic burden. A vaccine can significantly reduce the effect caused by RV in children with severe diarrhea. Is the only feasible method for the prevention and control of RV high morbidity and mortality [1-2].
Rotavirus particles consists of 3 layers: the outer capsid, internal and external capsid core. By VP7 and VP4.VP7 capsid formation of a relatively smooth surface, VP4 spikes, two are Neutralizing Antigen, can induce plays an important role in virus neutralizing antibody.VP4 during infection, as a cell binding protein VP4 of rotavirus infection was enhanced after cleavage. Therefore, in order to rotavirus coat protein gene VP4 gene was cloned into prokaryotic expression vector, to study the pathogenic gene expression in host strain, [3-5]. lay the foundation for the study of new oral vaccine
Objective: to construct a recombinant plasmid (pBEX/VP4) carrying the rotavirus coat protein VP4 gene, detect the expression of recombinant plasmid, and verify the safety of recombinant vector expressing protein.
Methods: (1) 34 pairs of primers were designed according to the gene sequence of mouse Genbank in VP4, VP4 gene was synthesized by overlapping PCR. On the basis of pGEX-5x-1, construct the shuttle expression vector pBEX, and recombinant VP4 into pBEX, the recombinant plasmid pBEX/VP4. (2) transformed Escherichia coli, the expression of SDS-PAGE protein to verify the expression of protein. The safety through intestinal loop experiments.
Results: (1) sequencing showed that the synthesized VP4 gene was correct and successfully constructed shuttle vector pBEX/VP4. (2) VP4 protein, which was successfully expressed in E.coli, and its expressed protein was proved to be non-toxic by intestinal loop test.
Conclusion: (1) in the absence of templates, overlapping PCR method can be used to synthesize the target gene; (2) the expression of VP4 protein in Escherichia coli, illustrate the construction of shuttle vector is successful, and the intestinal loop experiments verify the safety of protein expression, as will the recombinant shuttle vector into the host bacteria. As the research of new oral vaccine laid the foundation.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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