犬瘟热病毒YD株的分离鉴定及其H基因序列分析

从疑似犬瘟热(CD)病犬的脏器中分离到1株病毒,经间接免疫荧光试验、RT-PCR鉴定、红细胞凝集试验和对不同动物致病性试验,证实该病毒为犬瘟热病毒(CDV)强毒株,命名为CDV YD株。对H基因序列的测定和分析表明,YD株H基因与国内强毒株更接近,核苷酸同源性为98.4%~99.7%,氨基酸同源性为97.8%~99.7%,而与疫苗株核苷酸同源性较远为91.2%~91.5%,氨基酸同源性为90.3%~91.2%。推导的H蛋白氨基酸序列中有9个潜在的N连接糖基化位点(N-X-S/T),可能与病毒体外复制和中和抗体有关,另外有12个保守的半胱氨酸(Cys)残基,对H蛋白二级结构起重要作用;根据H基因核苷酸序列绘制的进化树表明,CDV YD株属于国内流行基因型:Asia-1型。该研究为了解当前中国CDV流行变异情况和犬瘟热疫苗的研发提供了数据。     

1株库蠓源版纳病毒的分离与全基因组序列分析

为分析云南省虫媒病毒的种类与遗传特征,在云南省师宗县采集库蠓进行病毒的分离与鉴定;通过全长cDNA扩增与高通量测序技术获取病毒全基因组序列,进行序列比对与系统发生树构建。结果显示,从采集的库蠓样本中分离出1株可在C6/36细胞上引起细胞病变的毒株（YNSZ043）,病毒基因组为分节段双链RNA,琼脂糖凝胶电泳呈"2-4-3"的带型特征;电镜观察可见直径为70~80 nm,呈"指环状",表面具有纤维突起的病毒粒子。全基因组测序结果显示,YNSZ043毒株为版纳病毒（Banna virus,BAV）,基因组大小为20 683 bp,由Seg-1（3 762 bp）至Seg-12（861 bp）12个基因节段组成,与中国BAV毒株各基因节段的核苷酸序列相似性在64.8%~99.6%之间,氨基酸序列相似性在58.8%~100%之间,在系统发生树上YNSZ043毒株与中国分离的BAV聚为一簇,形成独立的中国进化支系。对决定BAV基因型的Seg-12分析结果显示,YNSZ043毒株属于A2基因型,该毒株的Seg-5/VP5与越南分离BAV毒株的核苷酸和氨基酸序列相似性高达97.1%和97.6%,表明该毒株的Seg-5基因节段很可能与越南毒株之间发生了基因重配。研究结果丰富了中国BAV的基因组序列,为开展云南省BAV的流行病学研究提供了参考。     

Cloning and Sequence Analysis of HA and NA Genes of One Strain of H6N6 Subtype Avian Influenza Virus Isolated from Guizhou Province

To investigate the epidemic situation of H6N6 subtype avian influenza virus (AIV) in Guizhou province,A/duck/Guizhou/013/2014 was isolated from Sansui duck in live poultry market of Guizhou in 2014,the hemagglutinin (HA) and neuraminidase (NA) genes of DK/GZ/14 were subjected to clone and sequence analysis.The results showed that HA gene had the highest nucleotide homologies (97.5%) with the duck-origin H6N6 subtype AIV isolated from Eastern China in 2009,and the strains of HA gene proteolytic cleavage sites was P-Q-I-E-T-R-G,which accordeol with the molecular characteristic of low pathogenic AIV (LPAIV).However,NA gene of A/duck/Guizhou/013/2014 had the highest nucleotide homologies (98.2%) with the duck-origin H6N6 subtype AIV isolated from Fujian in 2007.The phylogenetic tree showed that A/duck/Guizhou/013/2014 and Hunan strains located in the same branch,while three duck-origin H6N6 subtype AIV isolated from Guizhou in 2007 and A/duck/Guizhou/013/2014 located in the different branch for HA and NA genes in genetic evolution,which suggested that A/duck/Guizhou/013/2014 was far with the local H6N6 subtype.The results also clearly indicated that duck-origin H6N6 subtype AIV had genetic diversity in duck population in Guizhou.     

Study on Length Polymorphism of KAP1 Family Genes in Yak (Bos gurnniens)

This study was aimed to understand the characteristics of length polymorphism with repeat sequence of keratin associated protein 1 (KAP1) family genes in yak. KAP1 family genes of yak and cattle were sequenced, and compared with sheep KAP1 family gene sequences. The results showed that cattle KAP1 family genes were located in chromosome 19, according to location of sheep KAP1 family genes in the chromosome and similarity with cattle KAP1 family genes, renaming the cattle KAP1 family (according to the gene location of chromosome) B2D, B2A, KAP1-1 and B2C genes into KAP1-4, KAP1-1, KAP1-2 and KAP1-3 gene, respectively. KAP1 family genes in the 3'and 5' flank were highly conserved, the difference between family genes mainly in the the repeat sequence region, which yak KAP1 to KAP4 genes were found 30 bp length polymorphism. There were B(CCQTS)A₁(CCQPT) repeat sequence and a new repeat sequence C(SIQTS). The results indicated that the repeat sequence was the key of the polymorphism of KAP1 family genes, which might be relate to combination with keratin protein.     

猪繁殖与呼吸综合征病毒分离株E11105 N基因的序列分析与原核表达

为研究PRRSV N蛋白的结构、功能以及N蛋白在病毒致病中的作用,以临床分离PRRSV毒株E11105为研究对象,采用Primer Premier 5.0设计一对特异性引物,经RT-PCR扩增出N基因片段,利用相关分子生物学软件对N基因序列进行分析;将N基因克隆连接到pColdⅠ原核表达载体上,经PCR、双酶切鉴定及序列测定后,得到重组质粒pColdⅠ-N,将pColdⅠ-N转入大肠埃希菌BL21(DE3)感受态细胞中,IPTG诱导表达后用SDS-PAGE蛋白电泳及Western blot验证分析。结果显示,分离株E11105N基因与北美洲型代表株VR-2332、欧洲型代表株Lelystad virus(LV)、中国2006年暴发的高致病性PRRSV代表株JXA1、中国代表株CH-1a的核苷酸序列同源性分别为93.3%、34.7%、99.2%、95.4%,氨基酸序列同源性分别为94.4%、15.3%、94.4%、99.2%;系统进化树显示,E11105株N基因与美洲型代表株VR-2332、中国高致病性JXA1毒株的亲缘关系较近;分离株E11105N基因所编码蛋白不存在跨膜区;二级结构主要以α-螺旋和无规则卷曲为主,分别占20.33%和63.41%;预测该蛋白可能存在5个较为明显的B细胞优势抗原表位。SDS-PAGE蛋白电泳结果表明,重组N蛋白主要存在于菌体沉淀中,分子质量约为16.7ku;Western blot结果显示,带His标签的重组表达蛋白能被His单克隆抗体识别,显色后条带约为16.7ku,与SDS-PAGE蛋白电泳的条带大小一致。     

Accuracy of genomic prediction using imputed whole‐genome sequence data in white layers

There is an increasing interest in using whole‐genome sequence data in genomic selection breeding programmes. Prediction of breeding values is expected to be more accurate when whole‐genome sequence is used, because the causal mutations are assumed to be in the data. We performed genomic prediction for the number of eggs in white layers using imputed whole‐genome resequence data including ~4.6 million SNPs. The prediction accuracies based on sequence data were compared with the accuracies from the 60 K SNP panel. Predictions were based on genomic best linear unbiased prediction (GBLUP) as well as a Bayesian variable selection model (BayesC). Moreover, the prediction accuracy from using different types of variants (synonymous, non‐synonymous and non‐coding SNPs) was evaluated. Genomic prediction using the 60 K SNP panel resulted in a prediction accuracy of 0.74 when GBLUP was applied. With sequence data, there was a small increase (~1%) in prediction accuracy over the 60 K genotypes. With both 60 K SNP panel and sequence data, GBLUP slightly outperformed BayesC in predicting the breeding values. Selection of SNPs more likely to affect the phenotype (i.e. non‐synonymous SNPs) did not improve the accuracy of genomic prediction. The fact that sequence data were based on imputation from a small number of sequenced animals may have limited the potential to improve the prediction accuracy. A small reference population (n = 1004) and possible exclusion of many causal SNPs during quality control can be other possible reasons for limited benefit of sequence data. We expect, however, that the limited improvement is because the 60 K SNP panel was already sufficiently dense to accurately determine the relationships between animals in our data.     

新城疫病毒La Sota株HN基因的克隆与序列分析

旨在从分子生物学角度进一步研究新城疫病毒La Sota株HN基因,探究NDV La Sota HN基因的遗传变异情况。研究以试验室冻存的p NDV-HN为模版,根据Gen Bank中登录的NDV La Sota株序列设计引物扩增HN基因,将其克隆到pMD18-T载体后进行鉴定,对鉴定正确的重组质粒p MD-NDV-HN进行测序分析。应用生物信息学软件对新城疫病毒Lasota株HN基因进行系统进化树分析、氨基酸序列同源性分析、糖基化位点、蛋白结构跨膜区分析及磷酸化位点预测分析。核苷酸序列测定结果表明:HN基因的序列长度为1743bp,该基因的开放阅读框(Open Reading Frame,ORF)总长为1716 bp,编码571个氨基酸。生物信息学表明:试验获得的HN基因具有6个潜在糖基化位点及12个半胱氨酸残基,第5个潜在糖基化位点(508-510 aa)发生缺失及第123位半胱氨酸残基被色氨酸残基取代。La Sota株HN基因编码的蛋白质中有29个丝氨酸、10个酪氨酸和16个苏氨酸可能成为蛋白激酶磷酸化位点。将试验获得的La Sota株HN基因序列和推导的氨基酸序列与新城疫毒株的HN基因相应序列比较后发现,它们的核苷酸序列同源性和氨基酸序列同源性最高均可达到99%,表明HN基因在种属间具有较高的保守性。研究为新城疫病毒的分子病毒学研究奠定了基础。