ALV實時熒光定量PCR檢測方法的建立及重組腺病毒介導(dǎo)的RNAi抑制ALV-J復(fù)制的研究
發(fā)布時間：2018-11-22 11:05
【摘要】：J亞群禽白血病病毒(subgroup J avian leukosis virus, ALV-J)是一種最常見的外源性禽白血病病毒,自1999年我國首次從商品代肉雞中分離得到之后,全國各地相繼報道了ALV-J的感染。ALV的感染可能會造成雞群生長遲緩、產(chǎn)蛋率和受精率等生產(chǎn)性能的降低、腫瘤發(fā)生,同時會造成感染雞的免疫抑制,給我國養(yǎng)雞行業(yè)造成了巨大的經(jīng)濟損失。然而目前還沒有一種藥物可以治療或者控制AL,除了凈化措施之外,也需要探索一種有效的抗病毒感染的方法。根據(jù)GenBank上ALV各亞群的三個主要編碼基因gag、pol、env中的保守序列設(shè)計了5對特異性引物,分別用于檢測蛋白酶PR、反轉(zhuǎn)錄酶RT、整合酶IN、表面蛋白基因gp85和穿膜蛋白基因gp37。通過RT-PCR技術(shù)擴增出5對引物的目的片段,回收PCR產(chǎn)物進(jìn)行克隆測序,測序正確后的樣品用于制備標(biāo)準(zhǔn)品質(zhì)粒,10倍系列連續(xù)稀釋后進(jìn)行SYBR Green Ⅰ實時熒光定量PCR,建立檢測的標(biāo)準(zhǔn)曲線,同時繪制熔解曲線。結(jié)果顯示,建立的5條標(biāo)準(zhǔn)曲線擴增效率為92.6%-100.5%,相應(yīng)的相關(guān)系數(shù)均超過0.994。經(jīng)過熔解曲線、特異性試驗結(jié)果分析,設(shè)計的5對引物只能擴增出ALV病毒,特異性好；敏感性試驗結(jié)果顯示,可以檢測得到一個數(shù)量級拷貝數(shù)的標(biāo)準(zhǔn)品質(zhì)粒。說明建立的方法敏感性高,可以用于檢測ALV-J病毒各基因mRNA轉(zhuǎn)錄水平的試驗。試驗通過構(gòu)建重組腺病毒介導(dǎo)的RNAi以研究其在DF。1細(xì)胞上抑制ALV-J毒株HG03病毒復(fù)制的能力,應(yīng)用建立的Real-time PCR方法檢測病毒PR、RT、IN、gP85和gP37基因的mRNA轉(zhuǎn)錄水平和囊膜蛋白gp85的表達(dá)水平,經(jīng)內(nèi)參GAPDH校正后計算出mRNA相對表達(dá)量,與對照組相比判斷其RNAi的抑制效果。首先根據(jù)GenBank中HPRS103毒株(Z46390.1)以及本實驗室所測J亞型毒株HG03 gp85基因保守區(qū)域為靶序列,遵循siRNA設(shè)計規(guī)則,以及載體pHBAd-U6-RFP的要求,設(shè)計了三對寡核苷酸siRNA1、siRNA2和siRNA3。為方便連接載體,序列加入EcoRⅠ和BamHI酶切位點。干擾序列連接腺病毒載體后,與骨架質(zhì)粒在HEK293細(xì)胞中進(jìn)行同源重組,獲得的3個重組載體分別命名為pAd-gp85-shRNA1\ pAd-gp85-shRNA2和pAd-gp85-shRNA3,空載體為pAd-N。經(jīng)測序鑒定正確后,制備重組病毒種子液并測定其滴度,分別達(dá)到1.26×1010PFU/mL、1.58×1010PFU/mL和1.26×1010PFU/mL,空載體滴度為2.0×1010PFU/mL。用構(gòu)建好的重組腺病毒在DF-1細(xì)胞上進(jìn)行RNAi效果的觀察。首先測定重組腺病毒感染DF-1細(xì)胞的最佳感染復(fù)數(shù)(the multiplicity of infection,MOI)為500。其次,用500 MOI的重組腺病毒感染長成單層的DF-1細(xì)胞,8h后按1×103TCID50的量接種HG03病毒,接種后48h分別用建立的SYBR Green Ⅰ實時熒光定量PCR方法檢測HG03各基因的mRNA相對轉(zhuǎn)錄水平。結(jié)果顯示構(gòu)建的重組腺病毒載體對HG03各基因都有抑制效果,對gp85基因抑制率為51.6%-82.0%,其中以pAd-gp85-shRNA2的抑制率最高,達(dá)82.0%；對其它4個基因的mRNA相對表達(dá)量也有所下調(diào),不同的基因下調(diào)的幅度不同。IFA檢測的結(jié)果顯示,干擾組的熒光強度明顯弱于陽性對照組的,而空載體對照組與陽性對照組的熒光強度沒有明顯區(qū)別,結(jié)果與Real-time PCR檢測的相符。本研究成功構(gòu)建了重組腺病毒介導(dǎo)的ALV-J HPRS103 gp85基因的RNAi載體,通過在DF-1細(xì)胞上進(jìn)行的干擾試驗結(jié)果證實,構(gòu)建的重組腺病毒干擾載體可有效抑制ALV-J分離株HG03在DF-1細(xì)胞上的復(fù)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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31
，
本文編號：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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.31
，
本文編號：2349161
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