猪细小病毒6型ORF 2基因的截短表达及多克隆抗体制备

【目的】原核截短表达猪细小病毒6型(Porcine parvovirus type 6,PPV6)ORF 2基因,并制备PPV6 VP1(348 aa-675 aa)蛋白多克隆抗体,为后续研究该蛋白的生物学功能提供材料。【方法】以PPV6分离毒株基因组为模板,PCR扩增获得ORF 2截短基因片段,将其克隆至原核表达载体pET30a(+)中构建重组质粒pET30a-PPV6-ORF2。经酶切和测序鉴定后,将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,Ni-NTA树脂亲和层析纯化,通过SDS-PAGE和Western blotting鉴定纯化后重组蛋白。将纯化后的重组蛋白与弗氏佐剂混匀乳化,免疫新西兰大耳白兔制备多克隆抗体,采用Western blotting、间接免疫荧光试验(IFA)和间接ELISA鉴定免疫兔血清特异性。【结果】成功构建了pET30a-PPV6-ORF2重组表达载体,原核截短表达了PPV6 VP1(348 aa-675 aa)蛋白;SDS-PAGE结果显示,重组PPV6 VP1(348 aa-675 aa)蛋白大小约为40 ku,主要以包涵体形式存在;Western blotting结果显示,该蛋白可与PPV6阳性猪血清发生特异性结合,具有良好的反应原性;Western blotting间接与ELISA结果显示,纯化后免疫兔血清与PPV6全病毒蛋白发生特异性反应,抗体效价为1∶25600;IFA鉴定结果表明,PPV6 VP1(348 aa-675 aa)蛋白兔源多克隆抗体具有良好的特异性。【结论】本研究成功原核截短表达了PPV6 ORF 2基因并制备了兔抗PPV6 VP1(348 aa-675 aa)蛋白多克隆抗体,为PPV6血清学检测方法的建立和PPV6 VP1蛋白的进一步研究提供了参考。

Expression of Porcine Circovirus Type 2 ORF2 Gene in Myocardial Cells of Detached Cherry Valley Duck

The objective of this study was to construct the recombinant eukaryotic expression plasmid of ORF2 gene harboring enhanced green fluorescent protein （EGFP） report gene. ORF2 gene of porcine circovirus type 2 cloned by PCR was ligated to the expression vector pEGFP-N1, which contains enhanced green fluorescent protein （EGFP） report gene, the recombinant eukaryotic expression plasmid pEGFP-N1-ORF2 was constructed successfully and was transfected into pre- pared duck myocardial cells （DMCs） by lipofectin. According to the result, the fluorescence expression was directly detected with fluorescence microscope, and the expression of ORF2 were analyzed by RT-PCR and indirect immunofluorescence assay （IFA） respectively. About 48 h after transfection, green fluorescent can be observed on transfected cells ： T-PCR and IFA were positive. This indicated that ORF2 gene of PCV2 was expressed efficiently in transfected duck myocardial cells.

猪圆环病毒Ⅱ型ORF2截短基因的克隆及原核表达

参照GenBank中猪圆环病毒Ⅱ型（Porcinecircovirus type 2，PCV2）的ORF2基因序列，设计合成1对引物，对PCV-2ZS株ORF2基因上含主要抗原位点的片段（ORF2-ME）进行PCR扩增，并克隆到pMD18-T Simple Vector中，进行序列测定。将重组质粒pMD—ORF2-ME的EcoR Ⅴ和Xhol Ⅰ双酶切产物插入原核表达载体pET-32a（＋），构建原核表达载体pET—ORF—ME。测序分析表明，克隆的ORF2-ME与GenBank上公布的PCV2毒株核苷酸序列同源性为99％～100％，推导的氨基酸序列同源性介于91％～100％之间。原核表达载体导入BL21（DE3）后用IPTG进行诱导表达，收集菌液进行SDS—PAGE和Western Blotting分析，结果表明ORF2-ME在BL21（DE3）中成功表达，所表达融合蛋白的相对分子质量约为40000，并能被PCV2阳性血清所识别，这就为PCV2感染诊断和抗体检测提供了依据。

PRRSV河南地方分离株ORF 2～6基因的克隆与序列分析

根据GenBank公布的猪繁殖与呼吸综合征病毒（PRRSV）ATCC VR-2332株的ORF2～6基因的核苷酸序列，设计合成4对特异性引物，用RT—PCR方法扩增出PRRSV河南地方株（命名为HN-2株）的ORF 2～6片段，克隆到pUC-57T载体并进行测序。应用DNAStar序列分析软件对分离株的ORF 2～6基因和其推导的氨基酸序列与不同来源的9个PRRSV毒株进行了同源性比较，结果表明：该分离株的ORF 2～6基因与不同来源美洲型毒株之间的同源性分别为89．1％～98．1％和81．4％～96．1％，而与欧洲型毒株之间的同源性仅为46．9％～61．9％和44．2％～45．0％；同时将分离株ORF 2～6基因的推导氨基酸序列与7个关洲型PRRSV毒株进行变异分析比较，证实了该分离株ORF 2～6基因发生了变异。

浙江地区猪圆环病毒2型检测及遗传变异分析

为了掌握浙江地区猪圆环病毒2型(porcine circovirus type 2,PCV2)流行病学特点和遗传变异情况,应用PCR检测方法,对2018年浙江地区猪场采集的临床疑似病料进行PCV2检测,并对部分阳性病料进行全基因扩增、克隆、序列测定和ORF 2遗传进化树分析。结果显示:329份临床疑似样品中,PCV2阳性率27.4%(90/329),其中宁波地区阳性率最高,为52%;保育猪和肥育猪阳性率最高,分别为38.8%(64/165)和68.8%(11/16)。选取7份病料进行全基因组扩增,每个毒株随机挑选3个或4个阳性克隆进行序列测定,共得到22个毒株进行遗传变异分析。22株病株同源性为93.3%~99.8%,与16个参考毒株同源性为93.5%~99.8%。根据遗传进化树分析,22个毒株分别处于3个分支,其中6株PCV2a,6株PCV2b,10株PCV2d,说明目前浙江地区PCV2d是优势毒株。ORF 2遗传进化树分析显示,ORF 2核苷酸同源性较全基因组核苷酸同源性低,3个分支分别处于PCV2b、PCV2d和PCV2e,预示浙江地区流行毒株可能在向PCV2e转变。本研究掌握了PCV2在浙江地区的流行动态,为浙江省PCV2疫苗株的选择和疫苗研发提供了理论依据。

Isolation and phylogenetic analysis of feline calicivirus strains from various region of China

Feline calicivirus(FCV)is an important feline pathogen mainly causing upper respiratory tract disease,conjunctivitis,and stomatitis,and it is classifed into genotype I and genotype II.To investigate the prevalence and molecular characteristics of FCV,this study collected 337 cat swab samples from animal hospitals in diferent regions of China from 2019 to 2021.The positive detection rate of FCV was 29.9%(101/337)by RT-PCR.Statistical analysis showed that FCV prevalence was signifcantly associated with living environment(p=0.0004),age(p=0.031)and clinical symptoms(p=0.00),but not with sex(p=0.092)and breed(p=0.171).The 26 strains of FCV were isolated using F81 cells.Phylogenetic analysis showed that 10 isolates belonged to genotype I,and 16 isolates belonged to genotype II.These 26 isolates were highly genetically diverse,of which HB7 isolate had three same virulence-related amino acid loci with VSD strains.Potential loci distinguishing diferent genotypes were identifed from 26 isolates,suggesting the genetic relationship between diferent genotypes.In addition,selection pressure analysis based on capsid protein of 26 isolates revealed that the protein is under diversifying selection.This study reveals the genetic diversity of FCV and provides a reference for the screening of vaccine candidate strains and the development of vaccines with better cross-protection efects.

吉林省猪圆环病毒3型的分子流行病学调查及遗传演化分析

为了解猪圆环病毒3型(porcine circovirus type 3,PCV3)在吉林省的流行情况和分子生物学特性,本研究通过PCR方法对吉林省2015-2017年的484份血清样品进行PCV3检测,将PCV3检测阳性的样品进行ORF2基因扩增和测序,并利用生物信息学软件DNAStar和Mega 6.06对ORF2基因的分子生物学特性进行分析。结果显示,吉林省2015-2017年PCV3样品总感染率和猪场感染率分别为28.1%(136/484)和65.8%(25/38),且呈逐年上升趋势。同源性分析结果表明,本研究获得的4株PCV3 ORF2基因的核苷酸和氨基酸序列同源性分别为98.3%～98.9%和97.7%～99.5%,4株PCV3 ORF2基因与国内外参考毒株ORF2基因的核苷酸和氨基酸序列同源性分别为97.7%～99.7%和96.7%～100%。遗传进化分析表明,PCV3存在2个亚群:PCV3a和PCV3b。本试验分离的PCV3毒株分别位于2个亚群上,1株属于PCV3a亚群,3株属于PCV3b亚群。PCV3毒株Cap蛋白第24(A、V)和27位(R、K)氨基酸的不同可能与PCV3毒株的进化相关。本试验结果表明,PCV3在吉林省猪群和猪场中存在很高的感染率,PCV3毒株之间高度保守,本研究结果为PCV3的分子特性研究提供了参考依据。