为构建一种能够用于开发猪细小病毒病毒样颗粒疫苗的猪细小病毒VP2蛋白的重组杆状病毒.根据GeneBank数据库中的猪细小病毒全基因组序列设计并合成了1对能扩增VP2基因的引物,采用PCR扩增猪细小病毒BJ-2株的VP2基因,并将其克隆到杆状病毒转移载体pFastBacH TA中,获得阳性质粒pFastBac-PPV VP2;测序后将该质粒转化到大肠杆菌DH10Bac感受态细胞中,获得含有VP2基因的重组杆状病毒质粒Bacmid-PPV VP2;最后将获得的重组杆状病毒质粒Bacmid-PPV VP2转染Sf9昆虫细胞,并成功拯救了能稳定表达猪细小病毒VP2蛋白的重组杆状病毒Bacmid-PPV VP2.结果发现,该重组病毒可用于稳定表达猪细小病毒VP2蛋白,并且表达的VP2蛋白可在体外自我组装形成病毒样颗粒,电镜观测其颗粒大小与猪细小病毒全病毒大小类似,且形成的病毒样颗粒与全病毒一样具有豚鼠红细胞凝集作用,凝集效价可达1:2048.故构建的表达猪细小病毒VP2蛋白的重组杆状病毒为研制猪细小病毒的基因工程亚单位疫苗奠定了基础.

Objective:To prepare and apply hepatitis C (HCV) RNA control material for proficiency testing in the laboratory, so as to evaluate laboratory testing capability for HCV RNA and improve detection quality.Methods:HCV RNA negative and positive human plasma samples were collected, and RNA was quantified by Hepatitis C virus nucleic acid (RNA) detection kit (fluorescence PCR). The quality control samples of 5 pieces per set (197101~197105) were prepared, including 3 pieces of HCV RNA positive samples and 2 pieces of HCV RNA negative samples. Ten tubes from each serial number of quality control products were randomly selected for homogeneity evaluation. The stability of each quality control product was evaluated after being stored at 37 ℃ for 1 to 7 days. The quality control product was applied in the proficiency testing of 14 clinical laboratories. The test results were collected and used to evaluate the ability of HCV RNA detection in different laboratories.Results:The homogeneity evaluation results indicated that no HCV RNA was detected in negative quality control samples numbered 197102 and 197104. For the positive controls numbered 197101, 197103 and 197105, the RNA quantitative value (Log10 value) CV% were 8.59%, 9.58% and 9.95%, respectively. The results of stability evaluation showed that HCV RNA was not detected in negative quality control samples numbered 197102 and 197104 after 1-7 days of storage at 37 ℃. For the positive controls numbered 197101, 197103 and 197105, the RNA quantitative value (Log10 value) CV% were 8.26%, 6.09%, 6.12%, respectively. The qualitative judgments of 5 quality control samples from 14 participating laboratories were consistent to the expected results, and the scores were all 100 points. The standard deviation I of the materials numbered 197101, 197103 and 197105 were -0.99 - 2.05, -1.16 - 1.84, -1.78 - 2.30, respectively.Conclusions:The HCV RNA control materials for proficiency testing in the laboratory were proved to have good homogeneity and stability. The application of HCV RNA control materials showed systematic or random errors in certain laboratories.

In January 2022, the 25-day-old Cherry Valley ducks from a Yangzhou farm were characterized by leg paralysis and had no characteristic pathological changes after autopsy. RT-PCR results showed that ducks were infected by Tembusu virus（TMUV）. In order to isolate the virus, the treated tissue samples were inoculated to 10-day-old duck embryos through the allantoic cavity and the duck embryo isolate virus was named YZ01. The results of phylogenetic analysis showed that the virus has a similar genetic evolution relationship with other waterfowl isolates in China, belonging to the clade of 2.2. To assess the pathogenicity of the isolate, 7-dayold Cherry Valley ducks were inoculated with intracerebral route. Three days post inoculation（dpi）, the ducklings showed difficulty in movement, tremor and other symptoms, and the mortality rate of ducklings reached 70% after 7dpi. The results showed that YZ01 strain was highly pathogenic to ducklings. The results of challenge protection experiment showed that the protection TMUV HB vaccine against challenge with YZ01 was 80%. This study provides epidemiological basis for TMUV infection.

以经典毒株C株的基因组为模板,测序后对NS3蛋白的二级结构进行分析,并对抗原表位进行预测,结果表明NS3的二级结构非常丰富并且存在丰富的B细胞抗原表位和T细胞抗原表位.截获3个不同的基因区域a、b和c,并设计相应的引物,分别扩增出约645、609和615 bp的靶基因,分别用pET-28a表达载体进行重组后,获得pET-28a-NS3a、pET-28a-NS3b与pET-28a-NS3c,用表达菌株BL21(DE3)进行表达,b段表达较高,可溶性表达较好,纯化后,可获得重组蛋白CSFV-His-NS3b,SDS-PAGE验证表达的重组蛋白大小约为27 kD,Western-Blot证实其具有良好的免疫原性,并且通过ELISA测得蛋白浓度约5 mg/mL,表明该蛋白特异性很强,可用于猪瘟诊断试剂盒的开发应用.

取实验室试制的安全性符合规定的3批次猪细小病毒杆状病毒载体灭活疫苗(rPP03株)分别免疫5～6月龄后备母猪、4～6月龄种公猪和经产母猪,免疫后不同时间采血,通过血凝抑制试验检测猪细小病毒(porcine parvovirus,PPV)血清抗体,观察抗体消长规律.根据试验结果确定该疫苗的免疫持续期和免疫程序:后备母猪5～6月龄免疫1次,2～3周后加强免疫一次;经产母猪于配种前1个月免疫一次;种公猪每6个月免疫一次,免疫持续期为6个月.

为了研究鹅星状病毒重组Capsid蛋白亚单位疫苗对种鹅的免疫效果,试验用鹅星状病毒重组Capsid蛋白亚单位疫苗对种鹅进行接种,并对种鹅所产蛋孵出的雏鹅进行攻毒,采用琼脂扩散试验法检测鹅血清中GoAs抗体的水平,记录雏鹅攻毒结果.结果显示:三批疫苗免疫28 d后试验组种鹅均有抗体产生;对照组种鹅的抗体均为阴性.试验组雏鹅攻毒后一切正常,存活率100％,无不良临床症状.对照组雏鹅攻毒后第3天精神沉郁、厌食、站立不稳、离群、排稀灰白色或黄绿色粪便,存活率50％,死亡鹅剖检可见关节肿大,关节内和内脏表面有石灰样尿酸盐沉积,肾脏肿大和充血,脾脏出血严重,有灰白色坏死灶.研究结果表明鹅星状病毒重组Capsid蛋白亚单位疫苗免疫效果良好,能有效预防鹅星状病毒的感染.

旨在研究重组鸡传染性法氏囊病毒VP2蛋白在多联疫苗中的免疫效果.将50羽21日龄SPF鸡平均分为5组,分别用新流法(新城疫ND-禽流感H9-传染性法氏囊IBD)三联疫苗进行免疫,1组为非免疫对照组,2组免疫剂量0.05 mL,3组免疫剂量0.1 mL,4组免疫剂量0.2 mL,5组免疫剂量0.3.免疫后第21天采血,检测ND/H9血凝抑制抗体和IBD琼脂免疫扩散抗体.并用传染性法氏囊病毒强毒BC6/85株F1代点眼攻毒,攻毒剂量为每只0.1 mL,观察96 h后剖检.结果表明,免疫后21 d,第5组血清中ND/H9 HI抗体分别为8.1log 2和9.0log 2,IBD抗体4个试验组均显著高于对照组(P<0.05),5组显著高于2组.1组攻毒鸡全部发病,法氏囊外观发黄,呈胶冻样,囊内剖开有黏液,部分肉眼可见出血.1组未攻毒对照和免疫组,均未见异常,免疫攻毒保护率为100％.结果表明,重组鸡传染性法氏囊病毒VP2蛋白在三联疫苗中免疫剂量为每只0.05 mL,具有良好的免疫保护力,且不影响疫苗中其他抗原的免疫效果.

猪感染猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)14 d后就可以产生针对非结构蛋白(Non-Structural Protein 7,Nsp7)的抗体,而且抗体水平很高,与N蛋白抗体水平相似,但比N蛋白抗体持续时间更长.另外,检测Nsp7抗体可用于区分PRRSV I型和II型的感染.本试验旨在建立一种快速、特异且敏感的检测血清中PRRSV抗体的方法.试验根据NCBI数据库录入的Nsp7蛋白序列(GenBank:EF536003)设计引物,利用聚合酶链式反应(Polymerase Chain Reaction,PCR)扩增Nsp7基因,构建原核表达载体pET28a-Nsp7,转化大肠杆菌BL21(DE3)感受态细胞,并诱导重组蛋白表达.利用亲和层析纯化重组蛋白,Western Blot鉴定表达产物.将纯化后的重组蛋白按不同浓度包被酶标板,通过方阵滴定法确定最佳抗原包被浓度及血清稀释度,并对其条件进行优化,最终建立检测PRRSV Nsp7抗体的间接酶联免疫吸附测定(Enzyme Linked Immunosorbent Assay,ELISA)方法.结果表明,试验表达了重组Nsp7蛋白,重组的Nsp7蛋白能与PRRSV阳性血清发生特异性反应,建立了一种基于重组Nsp7蛋白的间接ELISA检测方法.建立的间接ELISA检测方法分别与商品化PRRSV抗体检测试剂盒检测结果相比,两者符合率为87.74％.试验表明,建立的间接ELISA方法可以用于PRRSV抗体的检测.

[目的]开发和研制鉴别猪圆环病毒疫苗免疫和野毒感染的ELISA检测试剂盒.[方法]根据NCBI数据库录入的2型猪圆环病毒(porcine circovirus 2,PCV-2)REP蛋白序列(GenBank登录号为KX828581)设计引物,利用PCR扩增REP蛋白全长基,构建原核表达载体pET-28a-rep,转化大肠杆菌BL21(DE3)感受态细胞,并诱导重组蛋白表达.[结果]经过SDS-PAGE分析,获得了大小约40 ku的目的蛋白,与理论大小相一致.重组蛋白经镍柱纯化后重组蛋白浓度为40μg/mL,纯度约95％.Western Blot及ELISA鉴定结果显示,该重组蛋白具有良好的免疫原性和反应原性.[结论]鉴于该重组蛋白具有良好的免疫原性及反应原性,该重组蛋白可为开发区分猪圆环病毒2型疫苗免疫和野毒感染的检测试剂盒奠定基础.

为了建立起双抗体夹心ELISA定量测核衣壳蛋白(caspid protein,Cap)抗原方法,采用氯化铯密度梯度超速离心方法制备猪圆环病毒2型Cap蛋白标准品,制备出针对PCV2-Cap单克隆抗体及PCV2-Cap多克隆抗体,筛选最佳包被抗体和酶标抗体的稀释度,绘制标准曲线,验证方法,并应用该方法对不同转移载体构建的PCV2-Cap蛋白随时间变化的表达量进行测定.结果显示:最佳包被抗体和酶标抗体的稀释度均为1∶10 000,标准曲线的范围是15.6～250 ng/mL,线性相关系数≥0.99,准确性为(103.46±12.50)％,特异性良好;检测不同转移载体构建的Cap表达量随时间的变化,发现Cap含量接种后24h开始表达,72 h达到高峰,呈S形曲线增长模式.结果表明:已建立的双抗体夹心ELISA定量测Cap抗原方法灵敏度、精密性和准确性均符合研究要求.

The expression and immunogenicity of conserved domain of protein gD were researched for developing antibody detection kit of IBRV virus. Specific primers were designed for the amplification of the gene gD sequence with the immunoglobulin-like domain based on the full length gene gD sequence from bovine herpesvirus, type 1 (GenBank ID NO. NC_001847). The immunoglobulin-like domain gene of gD protein was inserted into plasmid pET-32a after PCR amplication, and was named as pET32a-ΔgD. The expression vector was then transferred into strain E. coli Rosseta (DE3) with aim to induce the recombinant protein. The result showed that 43000 recombinant proteins were obtained through the analysis of SDS-PAGE, and the relative molecular mass of them were accordance with the theoretical value of recombinant protein gD. After purified from nickel column, the concentration of them was 2.5 mg/mL, with the purity of about 95％. The recombinant proteins were proved of good immunogenicity after experienced the identification of Western Blot and Elisa, which indicated that they could be used as antigen for the development of antibody detection kit of IBR.

[Objective] To soluably express the VP2 protein of infectious bursal disease virus by using Escherichia coli system.[Method] Based on the vp2 full-length sequence of IBDV (GenBank ID No.AY704912), a pair of specific primers were designed for the amplification of the vp2 sequence which had been deleted the N-terminal 80 amino acid residues of VP2 protein.The truncation gene of VP2 protein of IBDV HQ strain was cloned, and inserted into plasmid pET-22b.After IPTG induction, the recombinant protein was expressed in soluble form in the periplasm of E.coli.[ Result] The recombinant protein was about 39 kDa as SDS-PAGE shown, and the purified recombinant protein had good immunogenicity as Western Blot shown.[ Conclusion] The IBDV VP2 protein could be effectively expressed in the periplasm of E.coli, and the recombinant VP2 protein showed good immunogenicity.

猪圆环病毒Ⅱ型(PCV2)属于单股DNA病毒,种属圆环病毒科,其致病性较强,且致病机理非常复杂.PCV2很少单独感染,经常与其他病原发生共感染且临床表现大多十分显著.PCV2疫苗已经在很大程度上控制了猪圆环病毒相关疾病(PCVAD)在北美和欧洲的发展[1].但该病仍在持续,造成全球性损害,特别是未经疫苗免疫的国家或地区. 猪呼吸与繁殖障碍综合征病毒(PRRSV)的病毒核酸为单股RNA且不分节段,种属动脉炎病毒科.该病可发生在各年龄阶段的猪群,其发病率和死亡率都非常高.母猪感染该病毒后可引起成年母猪繁殖障碍,而在仔猪身上主要表现为呼吸道疾病[2].