猪圆环病毒ⅡRep启动子功能分析及感染性克隆的构建
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摘要
【目的】猪圆环病毒(PCV)分两个血清型PCV1和PCV2。PCV2与猪群中发生的多种疾病有关,其中断奶仔猪多系统衰竭综合症(PMWS)危害最为严重,给各国养猪业造成了巨大损失。因此,探讨PCV2的致病机理对于PCV2相关疾病的防治具有重要意义。本研究通过分离株PCV2-DT接种健康仔猪尝试复制PMWS病例,以虫荧光素酶为报告基因进行PCV2的Rep、Cap启动子定位,Rep启动子上类干扰素刺激应答元件(vISRE)功能分析,构建PCV2感染性克隆及带HA-Tag的重组PCV2,为研究PCV2的体外增殖与致病性、致病机理以及疫苗研发、分子诊断研究等奠定基础。
【方法】(1)用PK-15细胞从东台市某猪场病料中分离获得PCV2-DT毒株,进行其基因组序列分析,并接种病毒于42日龄健康仔猪(无PCV1、PPV、PRV、PRRSV等感染)进行感染试验。(2)用Promotor Prediction软件预测PCV2-DT株的启动子位置,PCR扩增预测启动子片段后定向克隆于pGL3-Basic载体,构建重组载体pGL3-Bp1、pGL3-Bp2、pGL3-Bp3、pGL3-Bp4以及已知序列的PCV1 Rep启动子的重组载体pGL3-Bp5,各重组质粒分别转染PK-15细胞后,以Luciferase Assay System测定各预测启动子片段活性的有无及强弱。(3)将含PCV1和PCV2 Rep启动子的重组质粒pGL3-Bp5、pGL3-Bp2,以及对PCV2 Rep启动子上vISRE实施定点突变而构建的重组载体pGL3-Bpm,分别转染PK-15细胞,用不同剂量α-IFN刺激后测定启动子的活性变化。(4)将PCV2双拷贝基因正向串联克隆于载体pSK(+),构建重组质粒pSK-2PCV2,转染PK-15细胞,盲传4代后,用RT-PCR和IFA方法检测有无病毒粒子产生。(5)用SOE-PCR方法将HA-Tag基因插入到PCV2 Cap蛋白终止密码子前面,环化扩增的线性病毒基因组(Circo-DNA-HA)后转染PK-15细胞,盲传4代用RT-PCR、IPMA、扩增相应片段测序、血凝试验等方法进行重组PCV2-HA病毒粒子检测及其HA-Tag的稳定性检测。
【结果】(1)成功分离到PCV2-DT株,其与国内外35株分离株的同源性均在95%以上,仅存在一些点突变,由进化树可见PCV2各毒株之间没有明显的地域性。PCV2-DT株接种仔猪10d后呈现PMWS症状。(2)初步测定,PCV2 Rep启动子位于1705nt～1968nt,Cap启动子位于834nt～1309nt,且活性均高于对照组的SV40启动子。(3)α-IFN可使各启动子活性受到不同程度地抑制,PCV2 Rep启动子的活性与α-IFN剂量正相关,但不同剂量的α-IFN对PCV1 Rep启动子的活性影响不明显。PCV2 Rep启动子上vISRE实施突变后活性减弱,而且对α-IFN的作用不敏感。无论突变与否,高剂量的α-IFN均对Rep启动子具有极强的抑制作用。(4)pSK-2PCV2转染PK-15细胞,盲传4代后经RT-PCR、IFA检测有病毒粒子产生。(5)Circo-DNA-HA转染PK-15细胞,盲传4代后经RT-PCR、IPMA、克隆测序、血凝试验检测有带HA-Tag的重组PCV2-HA的病毒粒子产生,但该病毒不具有血凝活性。
【结论】(1)本次分离的PCV2-DT株与国内外35株分离株同源性均很高,仅存在一些点突变。PCV2-DT能够导致仔猪出现PMWS症状。(2)用虫荧光素酶为报告基因成功地对PCV2 Rep、Cap启动子进行初步定位。(3)α-IFN对PCV Rep启动子的活性具有抑制作用,通过vISRE对病毒复制可能具有调节作用。(4)含PCV2正向串联双拷贝基因组的重组质粒pSK-2PCV2具有感染性,转染PK-15细胞可产生病毒粒子。(5)Circo-DNA-HA具有感染性,转染PK-15细胞可获得带HA-Tag的重组PCV2-HA。
【Objective】Porcine circovirus contains two serotypes,PCV1 and PCV2.Some kinds of diseases are related with PCV2 in porcine groups.Especially,PMWS is the most serious and has made great economic losses in the word.So,researching the mechanism of pathogenesis of PCV2 is very significant for prevention and treatment of diseases which are related with PCV2.In this reseach,PCV2-DT was going to be isolated,and Rep and Cap promotors of PCV2 was going to be located,and the function of vISRE which lies in Rep promotor was going to be analyzed,and infectious clone of PCV2 and recombinant virus of PCV2 with HA-Tag were going to be constructed.This research would lay a foundation for studying multiplication in vitro,pathogenicity of PCV2, pathogenic mechanism,vaccine development,molecular diagnosis.
【Methods】(1) PCV2 was isolated from tissue of pig which came from DT city and died of PMWS by using PK-15 cell,and its genomic sequence was analyzed.Piglets of 42 days which were free of PCV1、PPV、PRV、PRSV were inoculated with PCV2-DT virus.(2) PCV2 promotors were predicted by using promoter prediction software and the sequences predicted were amplified by PCR and cloned into pGL3-Basic to construct pGL3-Bp1、pGL3-Bp2、pGL3-Bp3、pGL3-Bp4 as well as pGL3-Bp5 which contains known PCV1 Rep promoter.All recombinated plasmids were transfected into PK-15 cell,and the activity of promoters was detected by using Luciferase Assay System.(3) PGL3-Bp2 which contains PCV2 Rep promoter and PGL3-Bp5 which contains PCV1 Rep promoter and PGL3-Bpm which was constructed through mutating PCV2 Rep promoter in vISRE were respectively transfected into PK-15 cell.After being stimulated by different doseα-IFN,the changes of activity of promoters were detected.(4) Two copy gene of PCV2 was tandemLy cloned into pSK(+) to construct pSK-2PCV2.And pSK-2PCV2 was transfected into PK-15 cell,after 4 passages,the infected cell was detected by IFA and RT-PCR.(5) HA-Tag gene was cloned into Cap gene end by SOE-PCR.The PCV2 gene with HA-Tag was cyclized and amplied Then,circo-DNA-HA was transfected into PK-15 cell.After 4 passages,the cell was detected by RT-PCR、IPMA、Amplifying corresponding fragment and sequencing、HA experiment.Then,the stability of HA-Tag of the recombinated virus was detected by restriction enzyme NcoⅠdigestion.
【Results】(1) PCV2-DT is succeedly separated.Comparing with 35 domestic and abroad strains,the homology of nucleotide sequence are all over 95%,and only some sites mutated The phylogenetic tree demonstrates that there is no obvious regionalism between different strains of PCV2.After being inoculated with PCV2-DT for10 days,the piglets emerged the symptoms of PMWS.(2) PCV2 Rep promotor is located in 1705nt-1968nt;PCV2 Cap promotor is located in 834nt-1309nt.The activities of Rep and Cap promoter are more stronger than control SV40 promotor.(3) The activities of promotors are inhibited byα-IFN in different extent.The activity change of PCV2 Rep promoter is positively correlated with the dose ofα-IFN.But different doses ofα-IFN had no obvious influence on the activity of PCV1 Rep promoter.After vISRE being mutated,the activity of PCV2 Rep promoter weakens and becomes sensitive toα-IFN.Whether mutated or not,the activities of promotors were heavily inhibited by high doseα-IFN.(4) PCV2 is detected in PK-15 transfected with PSK-2PCV2 for 4 passages by RT-PCR and IFA.(5) Recombinant virus with HA-Tag(PCV2-HA) is detected in PK-15 transfected with Circo-DNA-HA for 4 passages by RT-PCR and IPMA,but PCV2-HA has no activity of adsorbing rhodocyte of chicken.
【Conclusions】(1) The homology between PCV2-DT and other 35 domestic and overseas strains was very high.PCV2-DT,and exists some sites mutated,and could induce piglet to emerge PMWS.(2) Rep and Cap promotor of PCV2 were firstly located by using luciferase as reporter gene(3) The activities of PCV promoters could be inhibited byα-IFN.Andα-IFN possibly regulates replication of virus wereby vISRE.(4) PSK-2PCV2 has infection activity,transfected into PK-15,could produce PCV2 virus.(5) Circo-DNA-HA has infection activity,transfected into PK-15,could produce recombinated PCV2 virus with HA-Tag.
【方法】(1)用PK-15细胞从东台市某猪场病料中分离获得PCV2-DT毒株,进行其基因组序列分析,并接种病毒于42日龄健康仔猪(无PCV1、PPV、PRV、PRRSV等感染)进行感染试验。(2)用Promotor Prediction软件预测PCV2-DT株的启动子位置,PCR扩增预测启动子片段后定向克隆于pGL3-Basic载体,构建重组载体pGL3-Bp1、pGL3-Bp2、pGL3-Bp3、pGL3-Bp4以及已知序列的PCV1 Rep启动子的重组载体pGL3-Bp5,各重组质粒分别转染PK-15细胞后,以Luciferase Assay System测定各预测启动子片段活性的有无及强弱。(3)将含PCV1和PCV2 Rep启动子的重组质粒pGL3-Bp5、pGL3-Bp2,以及对PCV2 Rep启动子上vISRE实施定点突变而构建的重组载体pGL3-Bpm,分别转染PK-15细胞,用不同剂量α-IFN刺激后测定启动子的活性变化。(4)将PCV2双拷贝基因正向串联克隆于载体pSK(+),构建重组质粒pSK-2PCV2,转染PK-15细胞,盲传4代后,用RT-PCR和IFA方法检测有无病毒粒子产生。(5)用SOE-PCR方法将HA-Tag基因插入到PCV2 Cap蛋白终止密码子前面,环化扩增的线性病毒基因组(Circo-DNA-HA)后转染PK-15细胞,盲传4代用RT-PCR、IPMA、扩增相应片段测序、血凝试验等方法进行重组PCV2-HA病毒粒子检测及其HA-Tag的稳定性检测。
【结果】(1)成功分离到PCV2-DT株,其与国内外35株分离株的同源性均在95%以上,仅存在一些点突变,由进化树可见PCV2各毒株之间没有明显的地域性。PCV2-DT株接种仔猪10d后呈现PMWS症状。(2)初步测定,PCV2 Rep启动子位于1705nt～1968nt,Cap启动子位于834nt～1309nt,且活性均高于对照组的SV40启动子。(3)α-IFN可使各启动子活性受到不同程度地抑制,PCV2 Rep启动子的活性与α-IFN剂量正相关,但不同剂量的α-IFN对PCV1 Rep启动子的活性影响不明显。PCV2 Rep启动子上vISRE实施突变后活性减弱,而且对α-IFN的作用不敏感。无论突变与否,高剂量的α-IFN均对Rep启动子具有极强的抑制作用。(4)pSK-2PCV2转染PK-15细胞,盲传4代后经RT-PCR、IFA检测有病毒粒子产生。(5)Circo-DNA-HA转染PK-15细胞,盲传4代后经RT-PCR、IPMA、克隆测序、血凝试验检测有带HA-Tag的重组PCV2-HA的病毒粒子产生,但该病毒不具有血凝活性。
【结论】(1)本次分离的PCV2-DT株与国内外35株分离株同源性均很高,仅存在一些点突变。PCV2-DT能够导致仔猪出现PMWS症状。(2)用虫荧光素酶为报告基因成功地对PCV2 Rep、Cap启动子进行初步定位。(3)α-IFN对PCV Rep启动子的活性具有抑制作用,通过vISRE对病毒复制可能具有调节作用。(4)含PCV2正向串联双拷贝基因组的重组质粒pSK-2PCV2具有感染性,转染PK-15细胞可产生病毒粒子。(5)Circo-DNA-HA具有感染性,转染PK-15细胞可获得带HA-Tag的重组PCV2-HA。
【Objective】Porcine circovirus contains two serotypes,PCV1 and PCV2.Some kinds of diseases are related with PCV2 in porcine groups.Especially,PMWS is the most serious and has made great economic losses in the word.So,researching the mechanism of pathogenesis of PCV2 is very significant for prevention and treatment of diseases which are related with PCV2.In this reseach,PCV2-DT was going to be isolated,and Rep and Cap promotors of PCV2 was going to be located,and the function of vISRE which lies in Rep promotor was going to be analyzed,and infectious clone of PCV2 and recombinant virus of PCV2 with HA-Tag were going to be constructed.This research would lay a foundation for studying multiplication in vitro,pathogenicity of PCV2, pathogenic mechanism,vaccine development,molecular diagnosis.
【Methods】(1) PCV2 was isolated from tissue of pig which came from DT city and died of PMWS by using PK-15 cell,and its genomic sequence was analyzed.Piglets of 42 days which were free of PCV1、PPV、PRV、PRSV were inoculated with PCV2-DT virus.(2) PCV2 promotors were predicted by using promoter prediction software and the sequences predicted were amplified by PCR and cloned into pGL3-Basic to construct pGL3-Bp1、pGL3-Bp2、pGL3-Bp3、pGL3-Bp4 as well as pGL3-Bp5 which contains known PCV1 Rep promoter.All recombinated plasmids were transfected into PK-15 cell,and the activity of promoters was detected by using Luciferase Assay System.(3) PGL3-Bp2 which contains PCV2 Rep promoter and PGL3-Bp5 which contains PCV1 Rep promoter and PGL3-Bpm which was constructed through mutating PCV2 Rep promoter in vISRE were respectively transfected into PK-15 cell.After being stimulated by different doseα-IFN,the changes of activity of promoters were detected.(4) Two copy gene of PCV2 was tandemLy cloned into pSK(+) to construct pSK-2PCV2.And pSK-2PCV2 was transfected into PK-15 cell,after 4 passages,the infected cell was detected by IFA and RT-PCR.(5) HA-Tag gene was cloned into Cap gene end by SOE-PCR.The PCV2 gene with HA-Tag was cyclized and amplied Then,circo-DNA-HA was transfected into PK-15 cell.After 4 passages,the cell was detected by RT-PCR、IPMA、Amplifying corresponding fragment and sequencing、HA experiment.Then,the stability of HA-Tag of the recombinated virus was detected by restriction enzyme NcoⅠdigestion.
【Results】(1) PCV2-DT is succeedly separated.Comparing with 35 domestic and abroad strains,the homology of nucleotide sequence are all over 95%,and only some sites mutated The phylogenetic tree demonstrates that there is no obvious regionalism between different strains of PCV2.After being inoculated with PCV2-DT for10 days,the piglets emerged the symptoms of PMWS.(2) PCV2 Rep promotor is located in 1705nt-1968nt;PCV2 Cap promotor is located in 834nt-1309nt.The activities of Rep and Cap promoter are more stronger than control SV40 promotor.(3) The activities of promotors are inhibited byα-IFN in different extent.The activity change of PCV2 Rep promoter is positively correlated with the dose ofα-IFN.But different doses ofα-IFN had no obvious influence on the activity of PCV1 Rep promoter.After vISRE being mutated,the activity of PCV2 Rep promoter weakens and becomes sensitive toα-IFN.Whether mutated or not,the activities of promotors were heavily inhibited by high doseα-IFN.(4) PCV2 is detected in PK-15 transfected with PSK-2PCV2 for 4 passages by RT-PCR and IFA.(5) Recombinant virus with HA-Tag(PCV2-HA) is detected in PK-15 transfected with Circo-DNA-HA for 4 passages by RT-PCR and IPMA,but PCV2-HA has no activity of adsorbing rhodocyte of chicken.
【Conclusions】(1) The homology between PCV2-DT and other 35 domestic and overseas strains was very high.PCV2-DT,and exists some sites mutated,and could induce piglet to emerge PMWS.(2) Rep and Cap promotor of PCV2 were firstly located by using luciferase as reporter gene(3) The activities of PCV promoters could be inhibited byα-IFN.Andα-IFN possibly regulates replication of virus wereby vISRE.(4) PSK-2PCV2 has infection activity,transfected into PK-15,could produce PCV2 virus.(5) Circo-DNA-HA has infection activity,transfected into PK-15,could produce recombinated PCV2 virus with HA-Tag.
引文
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