水貂阿留申病毒VP2重组蛋白与天然纯化抗原对比分析

发布时间：2018-06-23 17:33

本文选题：水貂阿留申病毒 + VP2基因　；参考：《吉林农业大学》2017年硕士论文

【摘要】：水貂阿留申病(Aleutian mink disease,AMD)是由阿留申病毒(Aleutian mink disease virus,AMDV)引起的一种慢性进行性传染病,是阻碍毛皮发展的三大传染病之一,AMD发病率约75%,直接死亡率可达30%。每年给水貂养殖业造成的经济损失难以估量。由于该病特殊的致病机理,目前还没有针对预防AMD的可靠药物,唯一的控制措施就是筛选并扑杀AMD阳性水貂。对流免疫电泳(CIEP)是公认检测AMD的金标准,其原理是抗原抗体在电场的作用下相向运动,两者相遇之后会形成一条清晰的白色沉淀线。形成沉淀线的为阳性,反之则为阴性。而抗原在检测AMD中具有重要的作用,其结果的准确性、反应的灵敏性主要由抗原的质量决定,因此抗原的研究和制备对AMD的检测工作有着重要的意义。本实验目的是筛选出一种成本低廉、操作便捷的抗原制备方法,为AMD的检测和AMD胶体金试纸条的制备提供基础。主要试验结果如下:通过对AMDV-G株的VP2基因序列进行原核表达,预计条带扩增大小为1944bp,从而构建原核表达质粒PMAL-c4x-VP2a,通过SDS-PAGE结果显示其分子量大小与文献报道一致;通过Western blot结果显示,该蛋白可以与AMD阳性血清特异性结合;对表达的蛋白进行直链淀粉树脂亲和层析纯化,从而得到麦芽糖结合融合蛋白,最终1 L的培养物可以得到80 mg的重组蛋白;根据GenBank发表的AMDV-G株全基因组序列,利用生物学软件对VP2基因主要抗原表位进行序列分析,设计并合成一对特异性引物,预计条带扩增大小为710 bp,与载体pEASY-Blunt Zero连接后,通过PCR、双酶切及序列测定,将鉴定正确的重组阳性质粒与原核表达载体PMAL-p5x连接,进而构建PMAL-p5x-VP2b原核表达质粒,通过SDS-PAGE显示其分子量大小为23 kDa;Western blot结果表明,该蛋白可以与AMD阳性血清特异性的结合;对表达的蛋白进行渗透休克法快速分离纯化,1 L的培养物最终可以得到120 mg的重组蛋白;利用Bac-to-bac表达系统对AMDV-G株的VP2全基因进行真核表达,将VP2基因与转移载体pFast BacHT-B连接,并转化到DH10Bac感受态细胞,通过与DH10Bac中的穿梭载体Bacmid发生转座,挑取白斑进行大量培养后提取转座子Bacmid-VP2,通过PCR鉴定后将正确的转座子Bacmid-VP2转染到生长至对数期的sf9细胞中,27℃培养箱培养72 h,收获部分病变的细胞并避光保存,同时对发生病变的细胞进行免疫荧光实验,结果显示,荧光信号强;通过病毒空斑实验对病毒筛选纯化;对表达的蛋白通过镍柱纯化后,1 L的细胞培养液可以获得100 mg的重组蛋白;对传统的AMDV抗原采用超滤管浓缩法进行纯化,其抗原纯度得到提高,将血清稀释至16倍后依旧可以检出,提高了检测的敏感性;利用生物学软件分析,原核表达系统中,PMAL-c4x-VP2a表达的目的蛋白占总蛋白的49.55%,而PMAL-p5x-VP2b表达的蛋白占总蛋白的71.07%;将三种重组蛋白抗原与AMDV抗原对临床上240个血清样本进行CIEP检测,通过对比,纯化的AMDV抗原其检测敏感性、特异性最好。而重组蛋白中,检出率最高的为真核表达的蛋白,与AMDV抗原相对比,其符合率达91.2%;而原核表达的两种蛋白,短片段表达的蛋白其敏感性及表达量都高于长片段所表达的蛋白,两者之间的符合率为85.3%;而短片段表达的蛋白与AMDV抗原的检出符合率达87.2%。
[Abstract]:Aleutian mink disease (AMD) is a chronic progressive infectious disease caused by Aleutian virus (Aleutian mink disease virus, AMDV). It is one of the three major infectious diseases that obstruct the development of fur. The incidence of AMD is about 75%. The direct mortality rate can reach to the inestimable economic loss caused by the mink breeding industry. The unique pathogenic mechanism of the disease has not yet been directed against the reliable drugs for the prevention of AMD. The only control measure is to screen and kill AMD positive mink. Convective immunoelectrophoresis (CIEP) is recognized as the gold standard for detecting AMD. The principle is that the antigen antibody is moving under the action of the electric field, and the two will form a clear white precipitate line after the two meet. The formation of the precipitate line is positive and the opposite is negative, and the antigen plays an important role in the detection of AMD. The accuracy of the results and the sensitivity of the reaction are mainly determined by the quality of the antigen. Therefore, the research and preparation of the antigen have important significance for the detection of AMD. The purpose of this experiment is to screen out a low cost and convenient operation. The method of antigen preparation provides the basis for the detection of AMD and the preparation of AMD colloid gold strip. The main results are as follows: by prokaryotic expression of the VP2 gene sequence of the AMDV-G strain, the size of the band amplification is 1944bp, and the prokaryotic expression plasmid PMAL-c4x-VP2a is constructed, and the molecular weight and the literature report of the SDS-PAGE result show its molecular weight and literature report. The Western blot results showed that the protein could be specifically combined with AMD positive serum, and purified the expressed protein by affinity chromatography of amylose resin to obtain maltose binding fusion protein, and the final 1 L culture could obtain a recombinant protein of 80 mg; according to the whole genome sequence of AMDV-G strain of GenBank published, the protein was used by GenBank. A pair of specific primers were designed and synthesized by biological software, and a pair of specific primers were designed and synthesized. The size of the band amplification was estimated to be 710 BP. After connecting with the carrier pEASY-Blunt Zero, the correct recombinant plasmid was identified with the prokaryotic expression vector PMAL-p5x through PCR, double enzyme digestion and sequence determination, and then the PMAL-p5x-V was constructed to construct PMAL-p5x-V. The prokaryotic expression plasmid of P2b showed that its molecular weight was 23 kDa by SDS-PAGE; Western blot results showed that the protein could be combined with the specificity of AMD positive serum; the protein expressed by osmotic shock was quickly separated and purified, and the culture of 1 L could eventually get a recombinant protein of 120 mg; the Bac-to-bac expression system was used for AMDV-G. The whole gene of VP2 of the plant is eukaryotic expression, the VP2 gene is connected with the transfer carrier pFast BacHT-B and transformed into the DH10Bac receptive cell, and the transposon Bacmid-VP2 is extracted from the shuttle carrier Bacmid in DH10Bac, and then extracts the transposon Bacmid-VP2 after a large amount of culture, and then the correct transposon Bacmid-VP2 is transfected to the growth. In the logarithmic phase of Sf9 cells, 72 h was cultured at 27 C incubator, and some cells were harvested and preserved. At the same time, the cells were immunofluorescent. The results showed that the fluorescence signal was strong; the virus was screened and purified through the virus empty spot experiment. After the purified protein was purified by the nickel column, the cell culture solution of 1 L could obtain 100. The recombinant protein of Mg was purified with ultrafiltration tube concentration for the traditional AMDV antigen. The purity of the antigen was improved. After the serum was diluted to 16 times, it could still be detected, and the sensitivity of the detection was improved. In the prokaryotic expression system, the target protein expressed by PMAL-c4x-VP2a accounted for 49.55% of the total protein, and PMAL-p5x-VP The protein expressed by 2B accounted for 71.07% of the total protein, and the three recombinant protein antigen and AMDV antigen were detected by CIEP in the clinical 240 serum samples. By contrast, the purified AMDV antigen has the best sensitivity and the best specificity. The highest detection rate in the recombinant protein is the eukaryotic expression protein, and the coincidence rate is 91.2% compared with the AMDV antigen. The sensitivity and expression of the protein expressed by the short fragment were higher than the protein expressed in the long fragment, and the coincidence rate between the two proteins expressed in the short fragment was 85.3%, and the coincidence rate of the protein expressed by the short fragment and the AMDV antigen was up to 87.2%..
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

【参考文献】