犬细小病毒病是由犬细小病毒(Canine paryovirus,CPV)引起的多发生于幼犬的致死性传染病,主要表现为出血性肠炎和心肌炎[1].CPV属于细小病毒科细小病毒属猫细小病毒亚群,其基因组为单股负链DNA.该病毒最早由美国学者A.K.Eugster等[2]于1977年从患肠炎的病犬中分离获得.此后,世界各国均有CPV感染家养犬及野生犬科动物的报道[3-4].田克恭等[5]建立了CPV分型鉴定的PCR方法并应用于实践.

为制备抗鸡新城疫病毒(NDV)血凝素-神经氨酸酶(HN)单链抗体(scFv)并鉴定其生物学功能,本研究提取抗NDV HN单克隆抗体杂交瘤细胞株总RNA,通过RT-PCR扩增抗HN蛋白抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其插入含有Linker序列的载体pET-scIG中,构建抗HN的scFv (HN-scFv)原核表达重组质粒pET-HN-scFv并在大肠杆菌中诱导表达.结果显示HN-scFv主要以包涵体形式存在.将包涵体进行复性和Ni-NTA层析柱纯化后用于流式细胞仪检测,结果显示复性纯化后的HN-scFv能够识别表达于293T细胞表面的NDV HN蛋白.该scFv的制备为进一步对该抗体进行改造奠定了基础.

The objective is to understand the complete genome and the genome organization of the porcine kobuvirus isolated in Fujian(PKV-FJ),China.Reverse transcription polymerase chain reaction were used to amplified the target gene fragments of the PKV-FJ,and the rapid-amplification of cDNA ends methods were employed to obtained the terminals for the virus using the 5′/3′RACE Kit(2nd generation).The full-genome of the PKV-FJ was 8 210bp including a single open reading frame(ORF)of 7 467bp encoding apolyprotein with 2 488amino acid.The 5′untranslated regions was 576bp long,while the3′untranslated regions was 167bp long.The polyprotein nucleotide sequences shared the highest identities with CH/HNXX-4/2012at 89.2%,and the lowest with swine/S-1-HUN/2007/Hungary at 87.6%,respectively.

The ORF1 Gene of porcine torque teno virus genogroup 2 was amplified by PCR method from the piglets sera genome association with post-weaning multisystem wasting syndrome from Fujian,the target product was cloned and sequenced.The results demonstrated that the porcine torque teno virus genogroup 2 ORF1 gene was 1875 bp in length,coding an open reading frame(ORF) with 624 amino acids.The nucleotide sequence and the amino acid sequence deduced from the gene were analyzed by the bioinformatics software.Compared with the porcine TTV2 ORF1 genes download from GenBank,the sequenced gene shared the highest homogeneity with FJ/China/2010/TTV2/2(GenBank accession number JF937656) strain at 98.8%,but shared lower homogeneity with SC strain(GenBank accession number HQ204188) strain only at 83.5%.According to the results of the phylogenetic relationship,the porcine torque teno virus had three subbranches,the Fujian isolates had two different branches,which means that there had more than two TTV2 sub-genogroups in Fujian,China.

A pair of specific primers targeted to the 3D gene of porcine kobuvirus was designed and a real-time reverse-transcription polymerase chain reaction(RRT-PCR) based on SYBR GreenⅠfluorescent was developed for quantization of the porcine kobuvirus infection.The standard curve generated a wide dynamic range of 6.42×102-6.42×108 DNA copies/μL with a linear correlation(R2) of 0.999 and efficiency of 100% between the Ct value and the logarithm of the plasmid copy number.The melting curve analysis using SYBR GreenⅠ dye showed one specific peaked with a melting temperature(Tm) of(84.94±0.24)℃ with no primer-dimer peak represent.No amplificon was detected from unrelated DNA samples by this method,such as porcine transmissible gastroenteritis virus,porcine rotavirus,porcine circovirus,porcine reproductive and respiratory syndrome,pseudorabies virus,and classical swine fever virus.Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.26%-1.14%% and inter-assay of 0.63%-1.79%.A real-time RT-PCR method established in this study may be used for earlier diagnosis and quantitative analysis of the infection status and the target organs infected with porcine kobuvirus.