为制备猪流行性腹泻病毒(PEDV)纤突糖蛋白(S)单克隆抗体,首先采用切向流超滤系统和凝胶过滤层析相结合的方法纯化PEDV CH/Hubei/2016毒株,免疫6～8周龄BALB/c小鼠,将血清抗体效价较高的小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,通过免疫过氧化物酶单层细胞试验(IPMA)对杂交瘤细胞株进行筛选,对阳性杂交瘤细胞进行亚克隆获得PEDV单克隆抗体杂交瘤细胞株.利用PEDV S1区域重组蛋白对单克隆抗体进行酶联免疫吸附试验和Western-blot鉴定,经间接免疫荧光试验(IFA)和激光共聚焦扫描显微术(CLSM)进一步验证单克隆抗体与病毒的反应原性.结果显示,所获得PEDV S1蛋白单克隆抗体共有4株,分别命名为9B9H6、10G11、17H6B10和18E9G6.通过小鼠体内诱生法制备腹水,抗体的IPMA效价在2.56×10-4至1.024×10-5之间.IFA结果表明,4株单克隆抗体均可与PEDV感染的Vero细胞发生特异性反应.进一步通过CLSM发现,4株单克隆抗体可以指示PEDV感染Vero 细胞过程中病毒新合成的S蛋白在内质网、内质网-高尔基体中间区域、高尔基体的亚细胞定位.PEDV S蛋白特异性单克隆抗体的成功制备,为PEDV感染机制研究和病原检测方法建立奠定了重要的分子基础.

为了分析猪德尔塔冠状病毒(PDCoV)、猪肠道α冠状病毒(PEAV)和猪流行性腹泻病毒(PEDV)在临床中的混合感染情况,采用反转录聚合酶链式反应(RT-PCR)方法将已经验证为PEDV阳性的样本进行PDCoV和PEAV检测.结果 表明,PDCoV和PEDV混合感染比例为6.28％,191份PEDV阳性样本中,PDCoV阳性为12份,PEAV阳性为1份,PDCoV、PEAV和PEDV共感染的比例为0.52％.结果 表明目前临床生产中PEDV仍然是引起仔猪腹泻的主要病毒性传染源,为临床生产中仔猪腹泻的防控工作提供理论依据和技术支撑.

为研究引起雏鹅痛风疫情的鹅星状病毒(Goose astrovirus,GAstV)河南流行株的基因组特征,从患有痛风的雏鹅样品中分离了1株GAstV,命名为XX株,并对分离毒株进行了全基因组测序和遗传特征分析.结果显示,GAstV XX株可在LMH细胞上稳定传代,但无明显的细胞病变;该毒株基因组全长7252 bp,与代表性毒株GD、AstV/SDPY/Goose/1116/17、AAstV/Goose/CHN/2017/SD01的基因组核苷酸序列同源性分别为98.1％、98.7％、98.7％.系统进化分析结果显示,GAstV XX株与GD、AstV/SDPY/Goose/1116/17、AAstV/Goose/CHN/2017/SD01等毒株处于同一进化分支,属于禽星状病毒1群.ORF2编码蛋白氨基酸序列分析结果显示,与已报道的GAstV流行毒株相比,GAstV XX株存在多个氨基酸位点的突变.

为研制灵敏、准确、稳定的猪瘟抗体检测试纸,对胶体金标记猪瘟病毒(CSFV) Ems-E2蛋白的pH、封闭液、保存液及样品垫等试纸生产各环节的保护剂、稳定剂及缓冲系统进行优化,确定猪瘟抗体检测试纸生产的工艺参数,使试纸的准确性、灵敏度及稳定性满足批量生产要求.该试纸在室温干燥条件下可稳定保存1 a以上,为实现猪瘟抗体检测试纸的商业化及广泛应用奠定基础.

为了得到一种能够高通量检测细胞培养物的方法,并将其用于筛选特异性强、敏感度高的抗猪繁殖与呼吸综合征病毒(PRRSV)单克隆抗体.用每孔能感染约100个细胞的病毒量接种单层覆盖96孔板的Marc-145细胞,12 h后用含3％H2 O2的甲醇固定细胞,以制备免疫过氧化物酶单层细胞试验(IPMA)反应板.以100μL/孔的量将融合后继续培养10 d的杂交瘤细胞的培养上清加入至IPMA反应板,以辣根过氧化物酶标记的羊抗鼠IgG-HRP作为二抗,以3-氨基-9-乙基咔唑(AEC)作为显色底物,于倒置显微镜下进行观察.结果表明,共筛选出1D1、7G8等39份PRRSV单克隆抗体,这39份单抗能够使PRRSV中高致病毒株HN07-1和经典毒株BJ-4感染的Marc-145细胞被特异性染色,而对猪瘟病毒、猪伪狂犬病毒、猪流行性腹泻病毒感染的Marc-145细胞无交叉染色.因此,构建的IPMA方法能够敏感、准确地捕捉到PRRSV单克隆抗体.

为了了解猪瘟病毒(CSFV)在PK-15细胞上的增殖规律.根据GenBank中CSFV Shimen毒株的NS5A基因保守区域序列,设计1对特异性引物,以CSFV总RNA为反转录模板,经优化反应条件,建立了基于实时荧光定量PCR技术的CSFV检测方法,并对其进行了敏感性、特异性、重复性试验,结果显示,该方法重复性好、特异性强,最低检测模板浓度为10拷贝/μL,并且CSFV的最低检测限为1TCID50/mL.利用所建立的方法研究CSFV在PK-15细胞上清中的增殖动态,并与TCID5o方法绘制的复制动态曲线进行比较.结果 显示,两种方法测定的CSFV在PK-15细胞上的复制动态具有一定的平行关系,该方法为研究猪瘟病毒的致病机理及利用体外细胞培养毒生产疫苗提供了试验依据.

为了研究甲基-CpG结合结构域蛋白2(MBD2)在体外对猪繁殖与呼吸综合征病毒(PRRSV)的复制是否有抑制作用.首先,构建MBD2的3'-UTR报告质粒,通过双荧光素酶报告结果表明MBD2是miR-373靶基因.然后用MOI=1的PRRSV感染MARC-145细胞,在24、36、48 h后分别取样,实时荧光定量PCR(qRT-PCR)和Western blots试验进行检测.结果表明,PRRSV感染下调MBD2的mRNA和蛋白的表达水平.用真核表达载体pcDNA3.1-Flag构建了MBD2的真核表达质粒pcDNA3.1-Flag-MBD2,用pcDNA3.1-Flag-MBD2转染MARC-145细胞,24 h后感染PRRSV,48 h后收获细胞.TCID50的试验结果表明,过表达MBD2能降低PRRSV滴度,而qRT-PCR和Western blots的结果则表明MBD2能降低PRRSV的载量;相反,MBD2的siRNA试验表明,下调表达有利于PRRSV在MARC-145细胞复制.通过基因缺失试验,构建MBD2部分结构域缺失的表达质粒,最终发现MBD2的GR与MBD结构域可能在MBD2抑制PRRSV复制起关键的作用.研究结果表明,MBD2是拮抗PRRSV的宿主内源性蛋白,并发现MBD2的GR与MBD结构域可能在MBD2抑制PRRSV复制中起关键的作用,而PRRSV可能利用miR-373来下调MBD2的表达,从而逃逸宿主对病毒的清除.

以真核质粒pcDNA3.1-flag-nsp1α为模板,利用PCR扩增出nsp1α基因片段,构建原核重组载体pET32a-nsp1α,转化到大肠杆菌BL21(DE3)感受态,1 mmol/L IPTG在37℃诱导表达6 h,成功获得以包涵体形式存在的重组蛋白,能够与小鼠抗His标签单克隆抗体反应;包涵体复性后作为抗原免疫新西兰大白兔,3次免疫后20 d获得兔多抗血清.通过间接ELISA、Western blot、间接免疫荧光、免疫组化等试验获得了高效价的兔多抗血清,该血清能够特异性地识别真核质粒表达的nsp1α以及PRRSV BJ4株.该试验成功表达了pET32a-nsp1α重组蛋白,并制备了高效价、高特异性的兔抗nsp1α多克隆抗体.

Specific primers were designed upon the conservative sequences of Marek's disease virus (MDV),infectious laryngotracheitis virus (ILTV),and fowlpox virus (FPV) genomic nucleotide sequences from registered in GenBank.Three pairs of specific primers for each virus were designed,a total of 9 pairs of primers for three viruses(MDV,ILTV,FPV) were screened by biological software,and three pairs of primers matched each other.After verification of three pair suitable primers with singleplex PCR,the reaction conditions of the multiplex PCR were optimized,including annealing temperature,concentrations of primers and Taq DNA polymerase.The simple multiplex PCR assay was successfully established after specificity,sensitivity and simplified test.The specific bands with lengths of 228 bp(MDV),400 bp (ILTV),499 bp(FPV) were amplified by the multiplex PCR,which were consistent with those expected.The results demonstrated that the method could simultaneously amplify these three viruses at a sensitivity of 100 fg when all three viruses were present.According to the principle of PCR technology,the classical PCR(a three step thermal cycle assay) was improved to be a two step thermal cycle assay,namely annealing and extension step were merged into one step (62 ℃),and the reaction time was shortened.This multiplex PCR for three DNA viruses was both highly specific and sensitive,and could be used as a rapid diagnostic assay for clinical samples.

To develop rice-derived subunit vaccine of Newcastle disease (ND),NDV F gene was screened,designed and synthesis after clone and construct recombinant plasmid pCAMBIA1300-F according to rice preference codon optimization.By constructing of vector twice,we made the target gene connect with vector pMP3,then transferred the gene to the vector pCAMBIA1300,finally Agrobacterium tumefaciens would be a mediate witch lead the gene to rice callus.The expressed rice seeds were got after screening by hygromycin.To obtain high expression of homozygous strain,the real time PCR were used to select 5 homozygous lines.The test of NDV-F antigen strip and Western blot indicated that the recombinant protein could react with good antigenic activity.This research would lay the foundation of the rice vaccine research.

依据Gen Bank中注册的禽流感病毒（AIV）、鸡传染性支气管炎病毒（IBV）、新城疫病毒（NDV）和传染性法氏囊病毒（IBDV）基因组核苷酸序列,通过序列比对获得AIV、IBV、NDV、IBDV的相对保守序列。根据每种病毒的保守序列利用生物学软件分别设计3对特异性引物,4种病毒共设计12对引物,并对12对引物进行模拟筛选,获得匹配最佳的4对引物。对获得的最佳匹配引物进行单项PCR验证,优化复合PCR反应条件,确定最佳的退火温度、引物浓度和Taq DNA聚合酶使用量;经特异性、敏感性及简化PCR试验,最终建立了简易复合PCR检测方法。结果表明,建立的检测方法可同时扩增出4条大小与设计相符的特异性片段,即IBV（146 bp）、NDV（215 bp）、IBDV（345 bp）、AIV（435 bp）;建立的复合PCR方法具有较强的敏感性和特异性,能检测出100 pg AIV、IBV、IBDV的cDNA和100 fgNDV的cDNA;将三温热循环改进为二温热循环,即将退火和延伸过程合为一步（62℃）,大大缩短了反应时间。

猪繁殖与呼吸综合征（PRRS）是严重危害养猪业的病毒性传染病之一,其致病原猪繁殖与呼吸综合征病毒（PRRSV）抑制宿主的天然免疫和特异性免疫反应,引起机体免疫抑制,造成持续性感染,从而给该病的防控带来困难。NLRP3炎症小体作为先天性免疫的重要组分在机体抗病毒中发挥重要作用。前期研究发现PRRSV能够激活NLRP3炎症小体,但PRRSV是否存在拮抗NLRP3炎症小体的组分还未见报道。本研究首先在缺失内源性炎症小体的HEK293T细胞中,共转染NLRP3、ASC、procaspase-1和pro-IL-1β四个真核表达质粒,建立NLRP3炎症小体的体外研究模型。然后,在该炎症小体模型细胞和猪肺泡巨噬细胞中,转染PRRSV nsp1α的真核表达质粒,结果表明nsp1α能够明显拮抗炎症小体的活化,而进一步的突变试验表明缺失N端锌指（ZF）结构或者突变ZF结构的nsp1α均不能抑制NLRP3炎症小体活化。本研究不仅首次发现了拮抗NLRP3炎症小体活化的PRRSV蛋白——nsp1α,而且发现nsp1α的N端锌指结构是其抑制NLRP3炎症小体活性所必需。本研究进一步发现了PRRSV拮抗天然免疫的新机制,并为PRRSV的防控提供了潜在的分子靶点和理论指导。

In order to obtain activated nonstructural protein 1α( nsp1α) of porcine reproductive and res-piratory syndrome virus (PRRSV),the nsp1α gene was amplified from the expression plasmid pcDNA3. 1-nsp1α of PRRSV BJ-4 strain and was subcloned into prokaryotic expression vector pET-28a. Then the pET-28a-nsp1α was transformed into E. coli BL21 (DE3). The E. coli BL21 (DE3) was induced by 1 mmol/L IPTG. SDS-PAGE result showed that the nsp1αwas mainly expressed in form of inclusion body and the molecular weight was 20 ku. Western blot result showed that nsp1αcould recognized with His-tag mouse Mcab. ELISA result showed that nspld could react with the PRRSV porcine positive serum which was diluted to 1∶6 400 . In this research the PRRSV nsp1α was expressed successfully and had good im- munological activity.

为研究靶向牛病毒性腹泻病毒(BVDV)的shRNA对BVDV复制的影响,利用脂质体介导法将BVDV shRNA真核表达质粒pSGH1-sh1083和pSGH1-sh8235及对照质粒pSGH1转染MDBK 细胞,通过G418抗性以及有限稀释法筛选获得BVDV shRNA单克隆细胞系subclonepSGH1-1083 和subclonepSGH1-8235及对照细胞系subclonepshmock.通过观察其细胞病变效应(CPE)、测定TCID50及RT-PCR、流式细胞术检测BVDV shRNA细胞系对BVDV复制的影响.结果表明,与阴性对照组相比,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235的细胞在感染BVDV 72 h后CPE不明显,其TCID50低于阴性对照组;RT-PCR结果显示,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235的细胞在感染BVDV后病毒E2基因的表达减弱;流式细胞术检测不同细胞系接毒24 h后的结合率,单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235与病毒结合率分别为11.37％和11.51％,与对照组(20.25％)相比有所降低.综上,稳定转染BVDV shRNA的单克隆细胞系subclonepSGH1-1083和subclonepSGH1-8235对BVDV的复制具有抑制作用.

目的:构建缺失免疫抑制位点的人早孕因子基因的原核表达载体pGEX6P-EPF11,诱导GST-EPF11融合蛋白在大肠杆菌中表达,并免疫小鼠,测定效价.方法:以pMD18T-HSPE1模板,PCR扩增缺失免疫抑制位点的人早孕因子基因片段,经限制性内切酶BamH I和Xho I双酶切后,连接到原核表达载体pGEX-6P-1中,构建重组表达质粒pGEX6P-EPF11.将表达质粒转化大肠杆菌BL21,以1 mmol/L IPTG 进行诱导表达GST-EPF11融合蛋白,Western blot鉴定.以表达的GST-EPF11融合蛋白作为免疫原免疫小鼠,抗体效价用ELISA检测.结果:在大肠杆菌中成功表达出相对分子质量约35 000的融合蛋白GST-EPF11.ELISA法检测免疫小鼠的抗体血清可达1:12 800.结论:在大肠杆菌中成功表达出GST-EPF11融合蛋白,并免疫了小鼠,测定了抗体效价,为下步制备缺失免疫抑制位点的人早孕因子的单克隆抗体(mAb)奠定了基础.

RT-PCR was used to study the expression of heat-shock 10 kDa protein.The results showed that there were two different HSP transcriptons of varied sizes in Homo liver cancer SMMC-7721 and Mus NS0.One HSP cDNA was composed of 312 bp(named HSPE1-1) and held 99% homology with HSPE1 mRNA reported(NM-002157).The other HSP gene cDNA was consisted of 313 bp(named Hspe1-1) and had 97% homology with Hspe1 mRNA reported(NM-008303).The homology between HSPE1-1 and Hspe1-1 was 92% in nucleotide sequence and 97% in amino acid sequence.Prokaryotic expression plasmids pET-28a-HSPE1-1 and pET-28a-Hspe1-1 for producing HSPE1 and Hspe1 proteins were constructed successfully.Its molecular weight was 14 kDa.Anti-His6 monoclonal antibody was used to demonstrate the identity of the proteins in Western Blot.The corresponding proteins were obtained after purification by Ni-NTA chromatography.The purified corresponding proteins would be helpfulin further studies of their molecular structure,biological function and clinical application.

The monoclonal antibodies(McAbs) against Glutathione-S-transferase(GST) were prepared in the present work.Balb/C mice were immunized with purified GST and the cell fusion was performed by standard method.The hybridom secreting antibodies were subcloned three times by limiting dilution.Two hybridomas,which were named as 4B7-E4 or 4B7-F4 and secreted McAbs against GST,were picked out by indirect ELISA.The ELISA antibody titers of cell culture supernatant and ascite of 4B7-E4 and 4B7-F4 were 1∶ 4 000 or 1∶ 3 000,and 1∶ 3×106 or 1∶ 2×106 respectively.Western-blot showed that these.McAbs can specifically react with GST and recombinant GST.

In order to produce the specific diagnostic antigens of classical swine fever virus(CSFV),the gene sequence of CSFV's main protective antigen-Erns protein was synthesized according to the sequence of the prevalent CSFV C-strain.Then the purified products were cloned into pPIC9K expression vector to construct recombinant expression plasmid(Ppic9k-Erns).After recombinant expression plasmid linearized by SalⅠ,the microzyme GS115 was transformed by SalⅠ.Through screening and identification,the positive recombinant microzyme of Erns protein to express CSFV was obtained successfully.This positive recombinant microzyme induced by methanol,then,the target protein could be examined in the supernatant culture solution.The results of Western-blot showed that this target protein could specifically recognize the antigens of CSFV and it had a great reactionogenicity.

目的:制备抗猪瘟病毒(CSFV)C株 E~(rns)/E2嵌合基因表达蛋白的单克隆抗体(mAb),探讨~(rns)/E2蛋白的分子结构和生物学功能.方法:在大肠杆菌 Rosetta~(TM)2(DE_3)中表达了猪瘟病毒(CSFV)C株 E~(rns)、E2蛋白主要抗原域的嵌合基因CSFV E~(rns)/E2蛋白;以纯化复性后的CSFV E~(rns)/E2为免疫原,按常规方法免疫BALB/c小鼠,应用淋巴细胞杂交瘤技术制备抗CSFV E~(rns)/E2 mAb;利用间接ELISA 、Western blot、Dot blot方法鉴定mAb生物学特性.结果:获得了1株稳定分泌抗CSFV Erns/E2融合蛋白的单克隆杂交瘤细胞系C8株,该株mAb的细胞培养上清效价为 1:6 400,小鼠腹水效价 1:2×10~5.其免疫球蛋白亚类为IgG1.间接ELISA和Western blot结果显示该株mAb不与猪瘟C株细胞毒、pET-32a标签蛋白反应,仅与融合表达的 CSFVE~(rns)/E2蛋白存在特异性反应.Dot blot结果表明其识别的抗原表位为E2蛋白的 RSGLCPFDTSPV氨基酸序列.结论:获得了一株能特异识别E2蛋白抗原表位的 mAb,为进一步研究 CSFV 的分子结构及功能奠定了物质基础.

The λ light chain of sIgM was cloned by RT-PCR from the total cellular RNAs of DT40 cells. DNA sequencing showed that the λ light chain of sIgM was 852 nt long including an 657 nt open reading frame (ORF) and 195 nt 3'-UTR. The sequence homology between DT40 sIgM λ light chain and chicken Ig λ light chain was 95 % at nucleotide level and 92.6 % in deduced amino acids. The PCR product was cloned into prokaryotic expression vector pET-28a for expression. SDS-PAGE analysis showed that the λ light chain of sIgM was abundantly expressed in E.coli BL21 (DE3). The present study provided foundation for further investigation on the role of sIgM in the infection process of IBDV to B-lymphocytes.