DHAV-3山东分离株P1基因序列分析及单克隆抗体的制备
发布时间：2018-09-07 11:06
【摘要】：鸭肝炎病毒(Duck hepatitis virus,DHV)导致的鸭病毒性肝炎(Duck viral hepatitis,DVH)以侵害三周龄以内的雏鸭为主,是一种急性高度致死性传染病。近年来,由3型鸭甲肝病毒(Duck hepatitis A virus,DHAV-3)引起的DVH广泛流行于中国和韩国,致死率高达80%,严重危害养鸭业的发展。为进一步了解山东省DHAV-3的流行近况、遗传变异规律和分子生物学特征,提高对该病的诊断速度和质量,加强对其的科学防控,本研究进行了以下两部分的研究:第一部分山东地区DHAV-3的病毒分离及分离株P1基因的序列分析2012年7月至2014年7月,对山东省四个地区(济南市、潍坊市、烟台市、临沂市)DHAV-3的流行情况进行了流行病学调查。共采集了18批167份疑似鸭病毒性肝炎致死的鸭肝组织等病料,经病料处理、鸭胚接种和雏鸭人工感染实验对病毒进行分离,用DHAV-3特异性检测引物对分离病毒进行RT-PCR扩增检测,共分离鉴定出70株DHAV-3野毒株。结果表明18批病料中均检出DHAV-3,批次检出率为100%;167份病料中有70份样品为DHAV-3阳性,个体阳性率为41.9%。从此次山东分离株中选取18株DHAV-3代表株,对其P1基因进行克隆测序和序列分析。同源性分析显示,18株DHAV-3分离株P1基因高度保守,其核苷酸和氨基酸序列同源性高达95.9%~99.8%和97.4%~100%。与27株DHAV-3参考株的核苷酸和氨基酸序列同源性分别为91.9%~99.0%和95.2%~99.6%,属同一血清型。进化树分析显示,中国分离株(除B63株)均属于GⅠ型。本研究分离的18株DHAV-3代表株均属于GⅠ型的S1亚型,其中济南、临沂、潍坊三地分离株遗传距离较近,位于同一小分支。越南分离株与中国B63株属于GⅡ型中的S3亚型,而所有韩国分离株组成GⅡ型中的S4亚型。DHAV-3的基因分型呈现明显的地域特征。氨基酸比对结果显示不同分支彼此之间存在多个氨基酸变异位点,其中共有22个氨基酸稳定变异位点。P1基因第671~712位氨基酸处于高变区(HVR),是病毒抗原变异的关键所在。在HVR内,所有的RGD序列变异为QSD序列。第二部分SD1101株单克隆抗体的制备为研制DHAV-3单克隆抗体,用已完成全基因组测序的SD1101株DHAV-3的全病毒免疫适龄BALB/c小鼠,取免疫效果最佳的小鼠的脾细胞和骨髓瘤细胞SP2/0进行融合,通过间接ELISA法对杂交瘤细胞进行筛选,经三次亚克隆后获得2株稳定分泌针对DHAV-3的单克隆抗体4A2和4C3。对这2株单抗的腹水效价、亚类、染色体数、特异性和稳定性进行了鉴定,结果如下:用间接ELISA检测连续传代和冻存复苏的细胞上清,发现其分泌抗体的能力无显著差异,证明其稳定性好;染色体数目在100左右,呈现典型的杂交瘤细胞染色体特征;经亚类鉴定,2株单抗分泌的抗体类型均为Ig G1亚类、κ亚型;单抗腹水效价为1:10240;IFA结果显示,2株单抗与DHAV-3和DHAV-1均发生特异性反应,但与DHAV-3的反应更强烈。
[Abstract]:Duck hepatitis virus (Duck hepatitis virus,DHV) caused by duck hepatitis (Duck viral hepatitis,DVH is an acute and highly fatal infectious disease, which mainly infects ducklings within three weeks of age. In recent years, DVH caused by duck hepatitis A virus type 3 (Duck hepatitis A virus,DHAV-3) is widely prevalent in China and South Korea. The fatality rate is as high as 80%, which seriously harms the development of duck breeding. In order to further understand the epidemic status, genetic variation and molecular biological characteristics of DHAV-3 in Shandong Province, improve the speed and quality of diagnosis of the disease, and strengthen the scientific prevention and control of the disease. The results of this study are as follows: the first part is the isolation of DHAV-3 virus and the sequence analysis of P1 gene in Shandong province from July 2012 to July 2014. Four regions of Shandong Province (Jinan, Weifang, Yantai) were studied. Epidemiological investigation on the prevalence of DHAV-3 was carried out in Linyi City. A total of 18 batches of 167 samples of duck liver tissue were collected. The virus was isolated by inoculation of duck embryo and artificial infection of ducklings. The virus was amplified by RT-PCR with DHAV-3 specific detection primers. A total of 70 DHAV-3 wild strains were isolated and identified. The results showed that the detection rate of DHAV-3, batches in 18 batches was 100 and 167. 70 samples were positive for DHAV-3, and the positive rate was 41.9%. Eighteen representative strains of DHAV-3 were selected from this Shandong isolate and their P1 genes were cloned and sequenced. Homology analysis showed that P1 gene of 18 DHAV-3 isolates was highly conserved, and the nucleotide and amino acid sequence homology was up to 99.8% and 97.4100% respectively. The homology of nucleotide and amino acid sequences with 27 DHAV-3 reference strains were 99.0% and 95.2%, respectively, belonging to the same serotype. Phylogenetic tree analysis showed that all Chinese isolates (except B63) belonged to G 鈪，
本文编号：2228085
[Abstract]:Duck hepatitis virus (Duck hepatitis virus,DHV) caused by duck hepatitis (Duck viral hepatitis,DVH is an acute and highly fatal infectious disease, which mainly infects ducklings within three weeks of age. In recent years, DVH caused by duck hepatitis A virus type 3 (Duck hepatitis A virus,DHAV-3) is widely prevalent in China and South Korea. The fatality rate is as high as 80%, which seriously harms the development of duck breeding. In order to further understand the epidemic status, genetic variation and molecular biological characteristics of DHAV-3 in Shandong Province, improve the speed and quality of diagnosis of the disease, and strengthen the scientific prevention and control of the disease. The results of this study are as follows: the first part is the isolation of DHAV-3 virus and the sequence analysis of P1 gene in Shandong province from July 2012 to July 2014. Four regions of Shandong Province (Jinan, Weifang, Yantai) were studied. Epidemiological investigation on the prevalence of DHAV-3 was carried out in Linyi City. A total of 18 batches of 167 samples of duck liver tissue were collected. The virus was isolated by inoculation of duck embryo and artificial infection of ducklings. The virus was amplified by RT-PCR with DHAV-3 specific detection primers. A total of 70 DHAV-3 wild strains were isolated and identified. The results showed that the detection rate of DHAV-3, batches in 18 batches was 100 and 167. 70 samples were positive for DHAV-3, and the positive rate was 41.9%. Eighteen representative strains of DHAV-3 were selected from this Shandong isolate and their P1 genes were cloned and sequenced. Homology analysis showed that P1 gene of 18 DHAV-3 isolates was highly conserved, and the nucleotide and amino acid sequence homology was up to 99.8% and 97.4100% respectively. The homology of nucleotide and amino acid sequences with 27 DHAV-3 reference strains were 99.0% and 95.2%, respectively, belonging to the same serotype. Phylogenetic tree analysis showed that all Chinese isolates (except B63) belonged to G 鈪，
本文编号：2228085
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