【作者】 张琳；

【导师】 姜世金；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2017， 硕士

【摘要】 鸭病毒性肝炎(Duck viral hepatitis,DVH)主要是由小RNA病毒科(Picornaviridae)禽肝病毒属(Avihepatovirus)的鸭甲肝病毒(Duck hepatitis A virus,DHAV)所引起的一种病程短、高度致死的传染性疾病。该病主要危害一月龄以内的雏鸭,对养鸭业造成巨大的经济损失。3C蛋白酶是小RNA病毒科病毒重要的蛋白酶之一,在病毒的复制过程中起到不可替代的作用。本研究利用实验室构建的鸭甲肝病毒1型(Duck hepatitis A virus type 1,DHAV-1)DNA-Lanched传染性克隆,通过PCR点突变的方法对3C基因的第38位和144位氨基酸进行定点突变,以研究突变后病毒的增殖能力及部分生物学活性的变化。本研究共分为三部分:1、3C蛋白的原核表达及抗血清的制备为了进行DHAV-1传染性克隆的IFA鉴定,本研究制备抗3C血清,首先将野生型3C基因及38位氨基酸突变基因3C38,144位氨基酸突变基因3C144,以及38和144位氨基酸同时突变基因3C38+144四组基因分别克隆到原核表达载体pGEX6P-1中,将鉴定正确的质粒转化到大肠杆菌BL21(DE3)中经诱导剂IPTG诱导表达出融合蛋白。将纯化后的融合蛋白与弗氏完全佐剂混合,免疫BALB/C小鼠,每只小鼠100μg。间隔两周后,使用同一免蛋白量相同量的融合蛋白与弗氏不完全佐剂混合,进行二免、三免和加强免疫。加强免疫结束后7天,小鼠眼球取血,离心后得到四份抗血清,ELISA检测发现四组抗血清中3C38的血清效价最高。2、3C突变基因传染性克隆的构建参照实验室保存的DHAV-1传染性克隆的序列和酶切图谱设计引物,分别扩增3C38、3C144、3C38+144和2C3A片段,通过引物将3C38、3C144、3C38+144与2C3A片段用EX Taq酶分别进行PCR融合,得到2C3A3C38、2C3A3C144和2C3A3C38+144片段,并连接到pMD18-T载体上,酶切鉴定得到阳性质粒,利用T载体通用引物进行测序,结果分析表明除人工诱变的定点突变外,其余部分与母本病毒中的3C序列的同源性均为100%。参考DHAV-1传染性克隆的酶切图谱,设计引物,将突变后的2C3A3C基因定向装配到全基因组中。首先将得到的2C3A3C均与3D片段进行融合,得到2C3A3C3D(文中简称为CD)片段,利用全基因组中存在的EcoRV酶切位点,将其按顺序装配到全基因组中,获得阳性重组子SR38、SR144和SR38+144,测序后发现只有定点突变的3C基因中的第38位氨基酸和第144位氨基酸发生突变,其余的氨基酸均无突变。将重组质粒SR38、SR144和SR38+144转染BHK-21细胞,以母本SR质粒作为阳性对照,以无菌的PBS作阴性对照。转染后,BHK-21细胞未出现明显的CPE。将转染后的四组细胞反复冻融后再次感染细胞,用荧光定量PCR和IFA方法检测均为阳性。经基因测序鉴定,表明病毒拯救成功。3、拯救病毒的增殖能力与部分生物学活性的检测重组质粒转染BHK-21细胞后,在细胞培养箱中培养48h后,收集细胞毒。用收集到的SR38、SR144和SR38+144三组拯救病毒与母本病毒再感染BHK-21细胞,并测定24h、36h、48h、60h和72h五个时间点病毒的拷贝数,绘制各病毒的生长趋势图。病毒的生长趋势图表明3C基因不同突变点的拯救病毒的增殖周期是不同的,并且病毒的最大拷贝数也是不同的,说明3C基因中的第38位氨基酸和第144位氨基酸的突变对病毒的增殖造成了一定的影响。为了研究拯救病毒的生物活性的变化,本研究将65枚9日龄的健康鸭胚分成五组。其中第1组向尿囊液中接种300μL的无菌PBS,余下的2、3、4、5组分别接种同剂量的拯救病毒SR、SR38、SR144和SR38+144。按24h、48h、72h、96h和120h五个时间点观察不同组鸭胚的临床症状,并收集尿囊液,利用荧光定量PCR检测病毒在其中的增殖情况。结果表明拯救病毒与母本病毒增殖周期与病毒拷贝数均存在差异,但在胚体上均可以观察到肝脏和脑出血、胚体上也具有明显的出血点等临床症状。更多还原

【Abstract】 Duck hepatitis A virus(DHAV),belonging to the Avihepatovirus of Picornaviridae,can cause duck viral hepatitis(DVH),which is a short duration,highly fetal infectious disease.DVH mainly threatens the ducklings within one month of age and brings huge economic losses in duck industry.3C protease is one of the important proteases of Picornaviridae virus,which plays an irreplaceable role in the replication process of virus.In this study,to determine the effects of amino acids mutations on viral proliferation capacity and partial biological activity,we mutated the amino acid at the position of 38 and 144 in 3C gene by PCR point mutation based on the DNA-Launched infectious clone of DHAV type 1(DHAV-1).1.Prokaryotic expression and preparation of antiserum of 3C proteinThe anti-3C serum was produced to carry out the IFA identification of DHAV-1 infectious clones in this study.Firstly,two amino acids sited in 38 and 114 were mutated alone and simultaneously in the wildly 3C gene,which was named 3C38,3C144 and 3C38+144 respectively.Prokaryotic expressions were performed by cloning the three mutations and the wildly 3C gene into vector pGEX6P-1.The positive plasmid was transformed into Escherichia coli BL21(DE3)to induce fusion protein induced by IPTG.Purified fusion protein was mixed with complete adjuvant of Freund’s to immunize BALB/C mice,each mice were inject 100 g.Second,third and strengthen immunization were performed using same quantity protein that mixed with incomplete adjuvant of Freund’s every two weeks.After seven days of strengthen immunization,antisera were taken from the blood of mice by centrifugation.The result of ELISA showed that the serum titer of 3C38 was much higher than these of the other three groups.2.Construction of 3C-mutation-gene infectious cloneThe,using the Designing primers based on the sequence and restriction map of DHAV-1 infectious clones stored in the laboratory to amplify 3C38,3C144,3C38+144 and 2C3 A.Fusing these fragments by PCR and constructed positive plasmid of 2C3A3C38,2C3A3C144 and 2C3A3C38+144 respectively.Sequencing results showed that those mutations shared 100% homology with the 3C sequence in the parent virus except artificial mutations.The mutated 3C gene was assembled to the whole genome referenced to the restriction map of DHAV-1 infectious clones.The 2C3A3C38,2C3A3C144 and 2C3A3C38+144 fragments were fused with 3D fragment to obtain 2C3A3C3D(hereinafter abbreviated CD)fragments with the mutations,which was assembled into the whole genome,using the EcoRV restriction site existing in the whole genome.Sequencing results showed that the mutations were found in38 th amino acid and 144 th amino acid in the site-directed mutagenesis of 3C gene and the rest of the amino acids were not mutated,meaning the positive recombinants SR38,SR144 and SR38+144 were obtained.The recombinant plasmids SR38,SR144,SR38+144 were transfected into BHK-21 cells,at the same time,parental SR plasmid was used as positive control while sterile PBS was used as negative control.We didn’t find any obvious CPE in BHK-21 cells after transfection while fluorescence quantitative PCR and IFA test showed positive.Gene sequencing indicated that the virus was rescued successfully.3.Detection of virus proliferation and the partial biological activity of the rescued virusBHK-21 cells were re-infected with SR38,SR144,SR38+144 groups and the female virus,collected after transfection of BHK-21 cells for 48 h.The growth trend of each saved virus was made according to the number of virus copies,measured at 24 h,36h,48 h,60h and 72 h.The growth trend of each rescue virus showed that both the proliferation cycle and the maximum copy number of the rescue virus that with different mutations in the 3C gene was different from the female virus,indicating that the mutation of the 38 th amino acid and the 144 th amino acid in the 3C gene had caused a certain impact on the proliferation of the virus.A total of 65 healthy duck embryos(9-day-old)were divided into five groups to study the biological diversity of the virus.A 300 μL sterile PBS was injected by allantoic cavity approach in the first group,Rescue virus of SR38,SR144,SR38+144 and the female virus SR were injected by same method respectively.Allantoic fluid was collected at the time of 24 h,48h,72 h,96h and 120 h to detect viral proliferation by QRT-PCR,at the same time,the clinical symptoms of duck embryos in different groups were observed.The result indicated that there were significant differences both in the proliferative cycle and virus c opy number between the saved virus and the female virus.Meanwhile,bleeding spots were clearly found in liver,brain and skin of embryos.更多还原