猪流行性腹泻病毒S1D蛋白的优化表达及其单克隆抗体的制备

为进一步探究猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)S蛋白的抗原表位及其功能,本试验通过优化S1D基因的密码子,构建了S1D基因未优化的重组原核表达质粒pET-S1D和已优化的pET-ΔS1D,并进行了诱导表达和纯化。使用SDS-PAGE和Western blotting方法验证S1D、ΔS1D蛋白在大肠杆菌内得到正确表达,利用Image J软件对S1D、ΔS1D蛋白表达量进行灰度扫描,通过t检验分析两者差异性。将纯化的ΔS1D 蛋白免疫 BALB/c小鼠,通过细胞融合、筛选及亚克隆,获得单克隆细胞株。利用体内诱生法制备抗PEDV S1D蛋白的单克隆抗体腹水,使用ELISA、Western blotting、间接免疫荧光试验3种方法对腹水效价及特异性进行检测和验证。SDS-PAGE和Western blotting结果显示,表达S1D、ΔS1D蛋白的样品均在34 ku处出现正确的目的条带。t检验结果表明, S1D、ΔS1D两者蛋白表达量差异极显著(P<0.01)。ELISA结果显示, 腹水的抗体效价达到了1∶1 000 000,腹水与PEDV病毒粒子和纯化后的ΔS1D蛋白反应均呈阳性,与PEDV N蛋白、pET-32a(+)空载体蛋白和猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)、猪瘟病毒(Classical swine fever virus,CSFV)、猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)和猪急性腹泻综合征冠状病毒(Swine acute diarrhea syndrome coronavirus,SADS-CoV) 5种病毒反应均呈阴性。Western blotting结果显示,腹水与ΔS1D蛋白和PEDV S蛋白分别在34和180 ku处有特异性条带出现,与pET-32a(+)空载体蛋白、正常Vero细胞蛋白均无特异性条带出现。间接免疫荧光试验结果显示,腹水及阳性对照组均能使细胞出现特异性绿色荧光信号,而空白及阴性对照组均未见绿色荧光信号。密码子优化可在原核表达系统中显著提高重组蛋白的表达水平,本研究基于高效表达的ΔS1D蛋白,成功制备了1株能稳定分泌与 PEDV S蛋白特异性结合的单克隆抗体的细胞株,为进一步探究 PEDV S蛋白抗原表位及蛋白功能的研究奠定了基础。     

猪戊型肝炎病毒ORF3蛋白影响HepG2细胞核黄素代谢的lncRNA-mRNA调控网络的鉴定

为初步鉴定并挖掘出猪戊型肝炎病毒(Hepatitis E virus,HEV)ORF3蛋白影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络,本试验通过构建腺病毒过表达载体,制备高滴度过表达腺病毒,介导猪 HEV-ORF3在HepG2细胞中实现过表达。Western blotting检测ORF3蛋白过表达成功后,运用lncRNA高通量组学测序,筛选出差异表达的lncRNA并进行靶向差异基因预测,对lncRNA靶向差异基因进行GO功能和KEGG通路富集分析,初步鉴定出与核黄素代谢信号通路相关的lncRNA-mRNA调控网络。Western blotting结果显示,在约为12 ku处出现目的条带,说明成功实现腺病毒介导猪HEV-ORF3在HepG2细胞中过表达。lncRNA高通量组学测序结果显示,共发现102个显著差异表达lncRNAs表达量上调,80个显著差异表达lncRNAs表达量下调。GO功能和KEGG通路富集分析显示,初步挖掘出lncRNA(MSTRG.13995.2)和lncRNA(MSTRG.1960.1)可能是与核黄素代谢信号通路相关的显著差异表达lncRNA,分别通过顺式调控其靶向基因APC-5和FLAD1来影响核黄素代谢信号通路。本试验初步鉴定出lncRNA(MSTRG.13995.2)-APC5和lncRNA(MSTRG.1960.1)-FLAD1可能是影响HepG2细胞核黄素代谢信号通路的lncRNA-mRNA调控网络。     

Study on the Change Rule of Egg Yolk Antibody in Laying Hens after First Immunization by Japanese Encephalitis Virus

The purpose of this experiment was to study the immunization rule of the egg yolk antibody affected by different vaccines,immunization dose and injection ways and further to discuss the optimal immunization procedures of the laying hens for the preparation of egg yolk antibody against swine Japanese encephalitis virus.180 brown laying hens without any vaccines were selected and divided into 18 groups randomly,each group of 10 hens.Groups 1,2 were the control groups,injected with the sterile saline;Groups 3 to 10 were injected with subcutaneous or intramuscular injection,and the vaccine was injected with 0.2,0.5,1.0 and 1.5 mL successively.Groups 11 to 18 were also adopted two kinds of injection,followed by the same dose of vaccine immunization.Six eggs of each experimental group were gathered before immune day and after 3,7,10,14,18,21 and 28 days,the egg yolk antibody was extracted and the titer was determined.As a result,the egg yolk antibody titers of groups 1 to 6,11 and 12 were all 0,and no significant immune response produced;The hens from 7 to 10 groups were injected with the inactivated vaccine.After 7 days,the average antibody titer reached the peak,and the duration of the antibody was 14 days.The hens from 13 to 18 groups were injected with the attenuated virus vaccine.After 14 days,the average antibody titer reached the highest value,and the duration of the antibody was 21 days.The egg yolk antibody titers were not significantly different in the two compared experiment groups with the same injection dose but with different injection ways (P>0.05).With the same injection way of each experiment group,and the difference was significant (P>0.05).Compared with some groups with the same injection and vaccine,the titer of yolk antibody was gradually increased with the increase of the immune dose,and the difference was significant (P<0.05).The results showed that,no matter intramuscular or subcutaneous injection,in order to produce a significant immune response to hens,the immune antigen dose was 1.0 mL inactivated vaccine or 0.5 mL attenuated vaccine at least.Compared with the attenuated and inactivated vaccine,inactivated vaccine stimulated the body to produce the antibody faster,but the maintenance time was shorter;The lower dose of attenuated vaccine could stimulate the body to produce antibodies,but the speed was slower,the maintenance time was longer.     

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.     

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.     

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.     

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.     

THORACIC RADIOGRAPHIC CHARACTERISTICS OF CANINE INFLUENZA VIRUS IN SIX DOGS

Canine influenza virus is an emerging, highly contagious, respiratory pathogen that has not previously been radiographically described. In this retrospective case series study, we describe the thoracic radiographic appearance of confirmed canine influenza virus in six dogs. Radiographic findings varied, but included abnormal unstructured interstitial (one) and unstructured interstitial and alveolar (five) pulmonary patterns, which were distributed cranioventral (four), diffuse (one), and caudodorsal (one). The right middle (five), left cranial (five), and right cranial (four) lung lobes were most commonly affected. Additionally, mild pleural effusion was present in one dog. Intrathoracic lymphadenopathy and cranial mediastinal widening/fluid accumulation were not detected in any dog. Canine influenza virus should be considered as a differential diagnosis for canine patients with respiratory signs and a cranioventral unstructured interstitial to alveolar pulmonary pattern.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.