本文選題：水貂阿留申病毒 + VP2基因��； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：水貂阿留申病(Aleutian mink disease,AMD)是由阿留申病毒(Aleutian mink disease virus,AMDV)引起的一種慢性進(jìn)行性傳染病,是阻礙毛皮發(fā)展的三大傳染病之一,AMD發(fā)病率約75%,直接死亡率可達(dá)30%。每年給水貂養(yǎng)殖業(yè)造成的經(jīng)濟(jì)損失難以估量。由于該病特殊的致病機(jī)理,目前還沒有針對預(yù)防AMD的可靠藥物,唯一的控制措施就是篩選并撲殺AMD陽性水貂。對流免疫電泳(CIEP)是公認(rèn)檢測AMD的金標(biāo)準(zhǔn),其原理是抗原抗體在電場的作用下相向運(yùn)動(dòng),兩者相遇之后會(huì)形成一條清晰的白色沉淀線。形成沉淀線的為陽性,反之則為陰性。而抗原在檢測AMD中具有重要的作用,其結(jié)果的準(zhǔn)確性、反應(yīng)的靈敏性主要由抗原的質(zhì)量決定,因此抗原的研究和制備對AMD的檢測工作有著重要的意義。本實(shí)驗(yàn)?zāi)康氖呛Y選出一種成本低廉、操作便捷的抗原制備方法,為AMD的檢測和AMD膠體金試紙條的制備提供基礎(chǔ)。主要試驗(yàn)結(jié)果如下:通過對AMDV-G株的VP2基因序列進(jìn)行原核表達(dá),預(yù)計(jì)條帶擴(kuò)增大小為1944bp,從而構(gòu)建原核表達(dá)質(zhì)粒PMAL-c4x-VP2a,通過SDS-PAGE結(jié)果顯示其分子量大小與文獻(xiàn)報(bào)道一致;通過Western blot結(jié)果顯示,該蛋白可以與AMD陽性血清特異性結(jié)合;對表達(dá)的蛋白進(jìn)行直鏈淀粉樹脂親和層析純化,從而得到麥芽糖結(jié)合融合蛋白,最終1 L的培養(yǎng)物可以得到80 mg的重組蛋白;根據(jù)GenBank發(fā)表的AMDV-G株全基因組序列,利用生物學(xué)軟件對VP2基因主要抗原表位進(jìn)行序列分析,設(shè)計(jì)并合成一對特異性引物,預(yù)計(jì)條帶擴(kuò)增大小為710 bp,與載體pEASY-Blunt Zero連接后,通過PCR、雙酶切及序列測定,將鑒定正確的重組陽性質(zhì)粒與原核表達(dá)載體PMAL-p5x連接,進(jìn)而構(gòu)建PMAL-p5x-VP2b原核表達(dá)質(zhì)粒,通過SDS-PAGE顯示其分子量大小為23 kDa;Western blot結(jié)果表明,該蛋白可以與AMD陽性血清特異性的結(jié)合;對表達(dá)的蛋白進(jìn)行滲透休克法快速分離純化,1 L的培養(yǎng)物最終可以得到120 mg的重組蛋白;利用Bac-to-bac表達(dá)系統(tǒng)對AMDV-G株的VP2全基因進(jìn)行真核表達(dá),將VP2基因與轉(zhuǎn)移載體pFast BacHT-B連接,并轉(zhuǎn)化到DH10Bac感受態(tài)細(xì)胞,通過與DH10Bac中的穿梭載體Bacmid發(fā)生轉(zhuǎn)座,挑取白斑進(jìn)行大量培養(yǎng)后提取轉(zhuǎn)座子Bacmid-VP2,通過PCR鑒定后將正確的轉(zhuǎn)座子Bacmid-VP2轉(zhuǎn)染到生長至對數(shù)期的sf9細(xì)胞中,27℃培養(yǎng)箱培養(yǎng)72 h,收獲部分病變的細(xì)胞并避光保存,同時(shí)對發(fā)生病變的細(xì)胞進(jìn)行免疫熒光實(shí)驗(yàn),結(jié)果顯示,熒光信號強(qiáng);通過病毒空斑實(shí)驗(yàn)對病毒篩選純化;對表達(dá)的蛋白通過鎳柱純化后,1 L的細(xì)胞培養(yǎng)液可以獲得100 mg的重組蛋白;對傳統(tǒng)的AMDV抗原采用超濾管濃縮法進(jìn)行純化,其抗原純度得到提高,將血清稀釋至16倍后依舊可以檢出,提高了檢測的敏感性;利用生物學(xué)軟件分析,原核表達(dá)系統(tǒng)中,PMAL-c4x-VP2a表達(dá)的目的蛋白占總蛋白的49.55%,而PMAL-p5x-VP2b表達(dá)的蛋白占總蛋白的71.07%;將三種重組蛋白抗原與AMDV抗原對臨床上240個(gè)血清樣本進(jìn)行CIEP檢測,通過對比,純化的AMDV抗原其檢測敏感性、特異性最好。而重組蛋白中,檢出率最高的為真核表達(dá)的蛋白,與AMDV抗原相對比,其符合率達(dá)91.2%;而原核表達(dá)的兩種蛋白,短片段表達(dá)的蛋白其敏感性及表達(dá)量都高于長片段所表達(dá)的蛋白,兩者之間的符合率為85.3%;而短片段表達(dá)的蛋白與AMDV抗原的檢出符合率達(dá)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%..
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65

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