ALV实时荧光定量PCR检测方法的建立及重组腺病毒介导的RNAi抑制ALV-J复制的研究
发布时间：2018-11-22 11:05
【摘要】：J亚群禽白血病病毒(subgroup J avian leukosis virus, ALV-J)是一种最常见的外源性禽白血病病毒,自1999年我国首次从商品代肉鸡中分离得到之后,全国各地相继报道了ALV-J的感染。ALV的感染可能会造成鸡群生长迟缓、产蛋率和受精率等生产性能的降低、肿瘤发生,同时会造成感染鸡的免疫抑制,给我国养鸡行业造成了巨大的经济损失。然而目前还没有一种药物可以治疗或者控制AL,除了净化措施之外,也需要探索一种有效的抗病毒感染的方法。根据GenBank上ALV各亚群的三个主要编码基因gag、pol、env中的保守序列设计了5对特异性引物,分别用于检测蛋白酶PR、反转录酶RT、整合酶IN、表面蛋白基因gp85和穿膜蛋白基因gp37。通过RT-PCR技术扩增出5对引物的目的片段,回收PCR产物进行克隆测序,测序正确后的样品用于制备标准品质粒,10倍系列连续稀释后进行SYBR Green Ⅰ实时荧光定量PCR,建立检测的标准曲线,同时绘制熔解曲线。结果显示,建立的5条标准曲线扩增效率为92.6%-100.5%,相应的相关系数均超过0.994。经过熔解曲线、特异性试验结果分析,设计的5对引物只能扩增出ALV病毒,特异性好；敏感性试验结果显示,可以检测得到一个数量级拷贝数的标准品质粒。说明建立的方法敏感性高,可以用于检测ALV-J病毒各基因mRNA转录水平的试验。试验通过构建重组腺病毒介导的RNAi以研究其在DF。1细胞上抑制ALV-J毒株HG03病毒复制的能力,应用建立的Real-time PCR方法检测病毒PR、RT、IN、gP85和gP37基因的mRNA转录水平和囊膜蛋白gp85的表达水平,经内参GAPDH校正后计算出mRNA相对表达量,与对照组相比判断其RNAi的抑制效果。首先根据GenBank中HPRS103毒株(Z46390.1)以及本实验室所测J亚型毒株HG03 gp85基因保守区域为靶序列,遵循siRNA设计规则,以及载体pHBAd-U6-RFP的要求,设计了三对寡核苷酸siRNA1、siRNA2和siRNA3。为方便连接载体,序列加入EcoRⅠ和BamHI酶切位点。干扰序列连接腺病毒载体后,与骨架质粒在HEK293细胞中进行同源重组,获得的3个重组载体分别命名为pAd-gp85-shRNA1\ pAd-gp85-shRNA2和pAd-gp85-shRNA3,空载体为pAd-N。经测序鉴定正确后,制备重组病毒种子液并测定其滴度,分别达到1.26×1010PFU/mL、1.58×1010PFU/mL和1.26×1010PFU/mL,空载体滴度为2.0×1010PFU/mL。用构建好的重组腺病毒在DF-1细胞上进行RNAi效果的观察。首先测定重组腺病毒感染DF-1细胞的最佳感染复数(the multiplicity of infection,MOI)为500。其次,用500 MOI的重组腺病毒感染长成单层的DF-1细胞,8h后按1×103TCID50的量接种HG03病毒,接种后48h分别用建立的SYBR Green Ⅰ实时荧光定量PCR方法检测HG03各基因的mRNA相对转录水平。结果显示构建的重组腺病毒载体对HG03各基因都有抑制效果,对gp85基因抑制率为51.6%-82.0%,其中以pAd-gp85-shRNA2的抑制率最高,达82.0%；对其它4个基因的mRNA相对表达量也有所下调,不同的基因下调的幅度不同。IFA检测的结果显示,干扰组的荧光强度明显弱于阳性对照组的,而空载体对照组与阳性对照组的荧光强度没有明显区别,结果与Real-time PCR检测的相符。本研究成功构建了重组腺病毒介导的ALV-J HPRS103 gp85基因的RNAi载体,通过在DF-1细胞上进行的干扰试验结果证实,构建的重组腺病毒干扰载体可有效抑制ALV-J分离株HG03在DF-1细胞上的复制。
[Abstract]:The sub-group J aviian leukosis virus (ALV-J) is one of the most common exogenous avian leukosis viruses, and the infection of ALV-J has been reported throughout the country since the first time in 1999 from the commercial broiler. The infection of ALV may cause the growth retardation of the chicken group, the production performance such as the laying rate and the fertilization rate, the tumorigenesis, and the immune suppression of the infected chicken, which has caused great economic losses to the chicken industry in China. However, there is currently no drug to treat or control the AL. In addition to the purification measures, a method of effective anti-viral infection is also required. Five pairs of specific primers were designed according to the conserved sequence of three major coding genes gag, pol, env of the ALV subpopulations on the GenBank, respectively for detecting the protease PR, the reverse transcriptase RT, the integrase IN, the surface protein gene gp85 and the membrane protein gene gp37. The target fragment of 5 pairs of primers was amplified by RT-PCR, and the PCR product was recovered for cloning and sequencing. The correct sample was used to prepare standard quality granules. After 10-fold serial dilution, SYBR Green I real-time fluorescence quantitative PCR was carried out to establish a standard curve for detection, and a melting curve was drawn. The results showed that the amplification efficiency of the five standard curves was 92.6%-100. 5%, and the corresponding correlation coefficient was more than 0.994. Through the analysis of the melting curve and the specific test results, the 5 pairs of primers designed can only amplify the ALV virus, and the specificity is good; the sensitivity test results show that the standard quality grains with an order of magnitude of the copy number can be detected. The method has high sensitivity and can be used for testing the mRNA transcription level of the ALV-J virus. By constructing the recombinant adenovirus-mediated RNAi to study the ability of the recombinant adenovirus to inhibit the replication of the ALV-J strain HG03 on the DF. 1 cell, the established Real-time PCR method is used for detecting the mRNA transcription level of the virus PR, RT, IN, gP85 and gP37 genes and the expression level of the envelope protein gp85, and the relative expression of the mRNA was calculated after the correction of the internal reference GAPDH, and the inhibition effect of the RNAi is judged in comparison with the control group. Three pairs of siRNA1, siRNA2 and siRNA3 were designed according to the design rules of siRNA and the requirements of vector pHBAd-U6-RFP. To facilitate the connection of the vector, the sequence was added to the EcoRI and BamHI sites. The recombinant vector was pAd-gp85-shRNA-1 pAd-gp85-shRNA 2 and pAd-gp85-shRNA 3, and the empty vector was pAd-N. After the sequencing and identification, the recombinant virus seed solution was prepared and the drop degree was determined, which reached 1.26-1010PFU/ mL, 1.58-1010PFU/ mL and 1.26-1010PFU/ mL, and the drop of empty carrier was 2.0-1010PFU/ mL. The effect of RNAi on DF-1 cells was observed with the constructed recombinant adenovirus. First, the optimal infection complex (MOI) of the recombinant adenovirus-infected DF-1 cell is 500. Secondly, a single-layer DF-1 cell was infected with the recombinant adenovirus of 500MOI, and the HG03 virus was inoculated in the amount of 1 to 103TCID50 after 8h, and the mRNA relative transcription level of the HG03 gene was detected by the established SYBR Green I real-time fluorescence quantitative PCR method. The results showed that the constructed recombinant adenovirus vector had the inhibitory effect on all the genes of HG03, the inhibition rate of gp85 gene was 51.6%-82.0%, and the inhibition rate of pAd-gp85-shRNA 2 was up to 82.0%, and the relative expression of the other four genes was also down-regulated, and the different genes were down-regulated. The results of IFA test showed that the fluorescence intensity of the interference group was significantly weaker than that of the positive control group, while the fluorescence intensity of the empty vector control group and the positive control group was not significantly different, and the result was consistent with the real-time PCR detection. The RNAi vector of the recombinant adenovirus-mediated ALV-J HPR103 gp85 gene was successfully constructed. The results of the interference test on the DF-1 cell confirmed that the constructed recombinant adenovirus interference vector could effectively inhibit the replication of the ALV-J isolate HG03 on the DF-1 cell.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
，
本文编号：2349161
[Abstract]:The sub-group J aviian leukosis virus (ALV-J) is one of the most common exogenous avian leukosis viruses, and the infection of ALV-J has been reported throughout the country since the first time in 1999 from the commercial broiler. The infection of ALV may cause the growth retardation of the chicken group, the production performance such as the laying rate and the fertilization rate, the tumorigenesis, and the immune suppression of the infected chicken, which has caused great economic losses to the chicken industry in China. However, there is currently no drug to treat or control the AL. In addition to the purification measures, a method of effective anti-viral infection is also required. Five pairs of specific primers were designed according to the conserved sequence of three major coding genes gag, pol, env of the ALV subpopulations on the GenBank, respectively for detecting the protease PR, the reverse transcriptase RT, the integrase IN, the surface protein gene gp85 and the membrane protein gene gp37. The target fragment of 5 pairs of primers was amplified by RT-PCR, and the PCR product was recovered for cloning and sequencing. The correct sample was used to prepare standard quality granules. After 10-fold serial dilution, SYBR Green I real-time fluorescence quantitative PCR was carried out to establish a standard curve for detection, and a melting curve was drawn. The results showed that the amplification efficiency of the five standard curves was 92.6%-100. 5%, and the corresponding correlation coefficient was more than 0.994. Through the analysis of the melting curve and the specific test results, the 5 pairs of primers designed can only amplify the ALV virus, and the specificity is good; the sensitivity test results show that the standard quality grains with an order of magnitude of the copy number can be detected. The method has high sensitivity and can be used for testing the mRNA transcription level of the ALV-J virus. By constructing the recombinant adenovirus-mediated RNAi to study the ability of the recombinant adenovirus to inhibit the replication of the ALV-J strain HG03 on the DF. 1 cell, the established Real-time PCR method is used for detecting the mRNA transcription level of the virus PR, RT, IN, gP85 and gP37 genes and the expression level of the envelope protein gp85, and the relative expression of the mRNA was calculated after the correction of the internal reference GAPDH, and the inhibition effect of the RNAi is judged in comparison with the control group. Three pairs of siRNA1, siRNA2 and siRNA3 were designed according to the design rules of siRNA and the requirements of vector pHBAd-U6-RFP. To facilitate the connection of the vector, the sequence was added to the EcoRI and BamHI sites. The recombinant vector was pAd-gp85-shRNA-1 pAd-gp85-shRNA 2 and pAd-gp85-shRNA 3, and the empty vector was pAd-N. After the sequencing and identification, the recombinant virus seed solution was prepared and the drop degree was determined, which reached 1.26-1010PFU/ mL, 1.58-1010PFU/ mL and 1.26-1010PFU/ mL, and the drop of empty carrier was 2.0-1010PFU/ mL. The effect of RNAi on DF-1 cells was observed with the constructed recombinant adenovirus. First, the optimal infection complex (MOI) of the recombinant adenovirus-infected DF-1 cell is 500. Secondly, a single-layer DF-1 cell was infected with the recombinant adenovirus of 500MOI, and the HG03 virus was inoculated in the amount of 1 to 103TCID50 after 8h, and the mRNA relative transcription level of the HG03 gene was detected by the established SYBR Green I real-time fluorescence quantitative PCR method. The results showed that the constructed recombinant adenovirus vector had the inhibitory effect on all the genes of HG03, the inhibition rate of gp85 gene was 51.6%-82.0%, and the inhibition rate of pAd-gp85-shRNA 2 was up to 82.0%, and the relative expression of the other four genes was also down-regulated, and the different genes were down-regulated. The results of IFA test showed that the fluorescence intensity of the interference group was significantly weaker than that of the positive control group, while the fluorescence intensity of the empty vector control group and the positive control group was not significantly different, and the result was consistent with the real-time PCR detection. The RNAi vector of the recombinant adenovirus-mediated ALV-J HPR103 gp85 gene was successfully constructed. The results of the interference test on the DF-1 cell confirmed that the constructed recombinant adenovirus interference vector could effectively inhibit the replication of the ALV-J isolate HG03 on the DF-1 cell.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
，
本文编号：2349161
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