化学佐剂对猪瘟病毒E2基因疫苗免疫增强作用的研究

以昆明小白鼠为试验动物，研究了化学佐剂对猪瘟病毒Ｅ１２囊膜糖蛋白（ＨＣＶ Ｅ２）基因疫苗的免疫增强作用；用印度墨水活体染色法筛选促进印度墨水在肌肉中扩散的化学试剂，并用筛选到的化学试剂与ｐＣＤＬａｃＺ混合肌肉注射小白鼠。结果表明，斯苯和甘油能较好的促进ＬａｃＺ基因在小白胫前肌中表达，提示斯苯和甘油可作为基因疫苗的化学佐剂；用斯苯和甘油按比例混合配制ＨＣＶ Ｅ２基因疫苗质粒ｐＣＤＳＴ免疫小白鼠，其抗     

猪瘟病毒E2基因疫苗表达抗原在小白鼠胫前肌的分布及消长

用免疫组化法检测了猪瘟病毒（ＨＣＶ）Ｅ２囊膜糖蛋白基因免疫表达的抗原在小白鼠胫前肌中的分布及消长，结果：ＨＣＶ Ｅ２基因疫苗ｐＣＤＳＴ接种小白鼠胫前肌后，表达的Ｅ２抗原主要分布于肌纤维膜上，少量分布于肌纤维间，在细胞浆中很难检测到Ｅ２抗原；用ＨＣＶ基因疫苗免疫后１ｄ，Ｅ２抗原已有表达，１３ｄ抗原达高峰，以后逐渐减少，３０ｄ仪检测到少量抗原。     

猪用转移因子对猪瘟免疫抗体影响的研究

为了研究猪用转移因子对猪瘟免疫抗体水平的影响,本研究选取25头20d龄仔猪进行了10周的抗体水平检测试验。试验猪随机分为试验Ⅰ、Ⅱ、Ⅲ、Ⅳ组和对照组,各组均在21d龄进行猪瘟疫苗首免,对照组用生理盐水稀释猪瘟疫苗肌肉注射,试验Ⅰ组用转移因子注射液0.2mL/头与用生理盐水稀释的猪瘟疫苗混合肌肉注射,试验Ⅱ组用转移因子注射液(0.2mL/头)与用生理盐水稀释的猪瘟疫苗分别肌肉注射,试验Ⅲ组在首免接种前3d,每天用转移因子注射液(0.2mL/头)肌注,第三天用生理盐水稀释猪瘟疫苗肌肉注射,试验Ⅳ组用生理盐水稀释猪瘟疫苗肌肉注射,同时用转移因子注射液(0.2mL/头)肌肉注射3d。各试验组和对照组分别于首免前1d,首免后第7d、14d、28d、42d、56d和70d由前腔静脉采血,采用猪瘟ELISA法检测猪瘟抗体水平。结果表明,与对照组比较,转移因子在猪瘟疫苗首免接种时或前后与猪瘟疫苗联合应用的免疫增强作用都是明显的,其增效作用的排序是Ⅲ组Ⅳ组Ⅰ组Ⅱ组。从本次试验结果看,转移因子可作为猪瘟疫苗的免疫增效剂,采用试验Ⅲ组的做法增效作用最好。     

鹅细小病毒VP3基因疫苗两种不同免疫方式对细胞免疫的影响

将构建的鹅细小病毒（GPV）VP3基因疫苗（pcDNA-GPV-VP3）转染鹅胚成纤维细胞（GEF），每隔12h检测VP3蛋白的表达情况，同时以不同剂量分别通过基因枪轰击和肌肉注射免疫28日龄健康雏鹅，于免疫后第3、7、14、21、28、35、49、63、77和105d采血，进行淋巴细胞增殖试验（MTT法）。结果，转染后第24h即可在GEF中检测到GPVVP3蛋白，第60h表达量达到高峰。试验组鹅外周血T淋巴细胞D490nm值在免疫后第35d达到最大。肌肉注射组和基因枪组免疫后第14～63d、第7～63d的D490nm值分别与PBS对照组差异板显著；肌肉注射组及基因枪组免疫后第21～49d的D490nm值显著高于弱毒疫苗对照组；肌肉注射组及基因枪组免疫后第14～63d的D490nm值板显著高于空质粒对照组。表明，基因枪轰击和肌肉注射pcDNA—GPV—VP3均能诱导雏鹅产生良好的细胞免疫应答。     

猪瘟病毒囊膜蛋白基因pcDNA4．0-E重组质粒的构建及其对小鼠的免疫原性

以猪瘟病毒石门株RNA序列为模板，应用RT—PCR方法，克隆得到猪瘟病毒囊膜蛋白E0-E2 cDNA全序列，并将其与真核表达质粒pcDNA4．0定向连接，构建了重组质粒pcDNA4．0-E。经提纯后，给试验组小鼠后肢胫前肌注射pcDNA4．0-E，每只100μg，15d后再注射1次，同时设空白对照组。于二免后第10、20、30d分别扑杀小鼠，进行免疫小鼠脾和外周血淋巴细胞的转化增殖试验，并用间接ELISA法检测血清抗体水平。结果显示，与空白对照组相比，所构建的重组质粒能够极显著增强小鼠脾细胞和外周血淋巴细胞的增殖（P〈0．01），血清抗体水平从二免后的第10d开始逐渐升高，且极显著高于对照组（P〈0．01）。说明该重组质粒能够诱导小鼠产生特异性免疫反应。     

种母猪免疫程序

以38d断乳母猪为例： 断乳前15～20d，补钙（维丁胶性钙）肌肉注射5mL；断乳前12～15d，伪狂犬疫苗臀部肌肉注射2mL。断乳前3～7d，猪瘟疫苗肌肉注射2头份；配种后60～70d．补硒，肌肉注射lOmL；配种后7l～80d：补钙（维丁胶性钙）5mL。配种后90～lOOd．补硒，肌肉注射10mL。临产前20～30d：流传二联苗后海穴注射4mL。临产前25～35d：萎鼻苗深部肌肉注射（指含巴氏杆菌毒素的）2mL。3、9、12月：肌肉注射口蹄疫疫苗3mL。根据断奶的早晚不同，再依据自己的实际情况应用。以上是建议性应用程序。     

猪白介素-4重组表达质粒在小鼠体内的组织分布及环境释放安全性

为了探讨pcDNA3.1/IL-4重组表达质粒作为疫苗佐剂进入临床应用的生物安全性,本试验就pcDNA3.1/IL-4重组表达质粒在小鼠体内的组织分布和环境释放安全性进行了研究.将pcDNA3.1/IL-4重组表达质粒经肌肉途径免疫BALB/c小鼠,免疫后不同时间剖杀动物并摘取各种组织,抽提基因组DNA.利用PCR技术分析其在组织内的分布及与细胞基因组发生整合的可能性,同时检测了免疫动物现场环境,以监测pcDNA3.1/IL-4重组表达质粒上的猪IL-4基因、CMV启动子基因和抗性基因是否在环境细菌中发生转移和扩散.结果表明,免疫后1 d在3/3小鼠各组织中仅注射部位肌肉存在重组表达质粒,免疫后5 d仅有1/3小鼠于注射部位肌肉中存在重组表达质粒,免疫后15 d所有小鼠的各组织中无重组表达质粒存在;未发现pcDNA3.1/IL-4重组表达质粒整合到宿主细胞基因组和转化到环境其他细菌中.因此,认为pcDNA3.1/IL-4重组表达质粒对动物和环境是安全的.     

捻转血矛线虫免疫调节型DNA疫苗的构建及在山羊体内表达研究

为构建免疫调节型DNA疫苗,将山羊IL-2基因分别与捻转血矛线虫H11-1、H11-2和H11-3基因串联构建融合表达载体pcDNA/IL-2-H11-1、pcDNA/IL-2-H11-2和pcDNA/IL-2-H11-3。为检测疫苗在山羊体内的表达情况,将纯化的疫苗质粒肌肉注射山羊后取注射部位肌肉组织,于首次免疫7 d后和二次免疫10 d后分别用RT-PCR和Western Blot等方法检测H11抗原的转录和翻译情况。结果发现H11基因和IL-2基因均能在注射部位肌肉获得转录和翻译。     

纹皮蝇HypoderminC基因真核表达质粒构建及小鼠免疫试验

根据GenBank发表的Hypodermin C(HC)基因序列设计引物,以原核表达载体pET-HC为模板,PCR扩增HC基因,将克隆基因插入到真核表达载体pVAX1中,构建重组真核表达质粒pVAX1-HC。将重组质粒pVAX1-HC转染小鼠胎儿成纤维细胞,以间接免疫荧光法检测HC基因在细胞中表达。用真核表达质粒pVAX1-HC经双侧胫前肌注射昆明小鼠,对小鼠进行体液免疫和细胞免疫研究。结果表明,小鼠血清抗体水平和淋巴细胞增殖水平明显高于非免疫对照组。本研究成功构建了真核表达质粒pVAX1-HC,为纹皮蝇核酸疫苗研发奠定了基础。     

昆虫细胞-杆状病毒系统分泌表达的猪瘟病毒E2蛋白及其免疫原性研究

为研制新型、可用于区分野毒感染、助力猪瘟净化的亚单位疫苗,本研究参考了近年来国内猪瘟病毒(CSFV)流行株的E2基因序列,并根据昆虫细胞密码子偏嗜性对其进行优化、合成后,克隆至转移载体中,构建了重组质粒,随后转化至含有穿梭质粒的大肠杆菌DH10Bac,制备了重组杆粒并转染至SF9细胞,获得了表达E2基因的重组杆状病毒。间接免疫荧光试验和western blot检测结果显示该杆状病毒感染昆虫细胞后可正确表达E2蛋白(Bac-E2)。将Bac-E2(50μg/头份)与猪瘟兔化弱毒疫苗[HCLV,7500半数兔体感染剂量(RID50)/头份]分别免疫猪瘟抗体阴性仔猪,3周后用相同的剂量加强免疫。首免后5周Bac-E2组猪CSFV中和抗体均不低于11 024,显著高于HCLV组(p0.01);首免后35 d,利用105.0MLD(最小致死剂量)的CSFV石门强毒经耳后颈部肌肉注射攻毒后,Bac-E2和HCLV组猪的保护率均为100%,阴性对照组猪全部发病、死亡;采用荧光定量PCR检测各组猪口腔和肛门拭子中CSFV的排毒情况,结果显示Bac-E2和HCLV组猪攻毒后0～16 d的咽拭子及肛拭子中均未检测到CSFV核酸。阴性对照组猪攻毒后第4 d至濒死前均可在其拭子中检出高拷贝CSFV核酸。本研究为Bac-E2蛋白作为猪瘟亚单位疫苗的候选抗原蛋白奠定了研究基础。     

Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV

The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.     

Incorporation of bovine bone marrow stromal cells into porcine foetal tissues after xenotransplantation

Bovine bone marrow stromal cells (BMSCs) were injected into the liver of foetal pigs at about 40 days of gestation to test whether these cells could populate developing tissue, and if so, which ones. Approximately 40 days after injection, the foetuses were harvested and tissue sections from many areas of the body were analysed for the presence of bovine cells using two different methods. First, using PCR, bovine repetitive DNA was found to be present in DNA extracted from foetal pig tissues. Secondly, using oligonucleotide primed in situ synthesis (PRINS), the in situ presence of bovine cells was found within porcine tissue sections. PRINS-labelled cells were found within cartilage, perichondrium, connective tissue and smooth muscle. These data suggest that bovine BMSCs integrate throughout the foetal pig.     

Development of polymerase chain reaction and comparison with in situ hybridization for the detection of Haemophilus parasuis in formalin-fixed, paraffin-embedded tissues

DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.     

猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响

采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应.     

ELISA法检测猪瘟抗体消长规律的试验研究

本试验采用间接ELISA方法,检测猪瘟(CSF)常规免疫仔猪的CSF抗体水平。试验分A、B两组,A组首免注射4头份猪瘟兔化弱毒疫苗,B组首免注射2头份猪瘟兔化弱毒疫苗;二免两组均注射2头份猪瘟兔化弱毒疫苗。试验结果表明,注射疫苗后10d,开始产生抗体,20d-30d抗体达到峰值,二免后抗体峰值维持40d以上。首免4头份的仔猪比首免2头份的仔猪CSF抗体峰值出现的早,维持时间长。     

Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.     

禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性

采用本实验室构建的3种禽传染性支气管炎病毒基因的真核表达质粒pIBVS1、pIBVM、pIBVN,按各50 mg/L配制成质量浓度为150 mg/L的DNA疫苗,经腿部肌肉分点注射免疫1周龄SPF雏鸡。分别在免疫后24 h,73、0及60d,采集试验鸡的血液、心、肝、脾、肺、肾、胸腺、性腺（卵巢/睾丸）及注射部位肌肉进行组织总DNA抽提,以组织总DNA为模板进行PCR扩增。另外,在对照组DNA模板中加入不同拷贝数的质粒,确定PCR反应的灵敏性。以纯化后的组织总DNA为模板,PCR法检测质粒DNA整合到鸡细胞染色体基因组上的可能性,评价疫苗的安全性。结果表明,该疫苗在24 h内迅速分布于全身,并能在血液及所有检测的组织器官内持续分布60 d;纯化后的组织基因组DNA经PCR扩增均呈阴性,未发现整合现象,证实该DNA疫苗的安全性好。     

猪瘟病毒E2-GM-CSF融合蛋白在HEK293T细胞中的表达和免疫原性分析

【目的】 设计构建分泌表达E2-GM-CSF融合蛋白的HEK293T重组细胞, 并评估表达的E2-GM-CSF融合蛋白的免疫原性, 为猪瘟病毒(Classical swine fever virus, CSFV)的防控提供新的候选亚单位疫苗。【方法】 利用PCR方法扩增E2-GM-CSF基因并克隆入慢病毒表达载体pCDH, 将得到的重组慢病毒质粒pCDH-E2-GM-CSF包装成E2-GM-CSF慢病毒颗粒, 转导HEK293T细胞, 经嘌呤霉素加压筛选获得分泌表达E2-GM-CSF融合蛋白的HEK293T重组细胞, 纯化表达的E2-GM-CSF融合蛋白经小鼠免疫试验验证其免疫原性。【结果】 pCDH-E2-GM-CSF质粒经酶切鉴定, 得到大小分别为8 172 bp的载体片段和1 521 bp的E2-GM-CSF基因片段, 表明E2-GM-CSF基因成功克隆入pCDH载体; 细胞基因组DNA PCR扩增结果为一条大小为1 686 bp的条带, 表明E2-GM-CSF基因成功整合至HEK293T细胞基因组; Western blotting分析获得约70 ku的条带, 证明E2-GM-CSF融合蛋白在HEK293T细胞中成功分泌表达; SDS-PAGE验证显示, 纯化后的E2-GM-CSF融合蛋白为单一条带, 纯度较高, 大小约70 ku; 小鼠免疫血清ELISA检测结果表明, 表达的E2-GM-CSF融合蛋白具有良好的免疫原性, 免疫后第14天在小鼠血清中检测到效价为1∶300的E2特异性抗体, 免疫后第21天E2特异性抗体效价达1∶900, 免疫后第28天小鼠血清中的E2特异性抗体效价最高达到1∶8 100, 远高于E2蛋白的1∶1 800。【结论】 利用慢病毒载体构建的表达E2-GM-CSF融合蛋白的HEK293T重组细胞可正确分泌表达具有免疫原性的E2-GM-CSF融合蛋白, 为CSFV的防控提供了新的候选亚单位疫苗, 同时其构建、开发与评估过程也可为其他亚单位疫苗在哺乳动物细胞中的快速表达提供借鉴。