猪传染性胸膜肺炎重组亚单位疫苗诱导小鼠免疫保护效果的观察

重组表达猪传染性胸膜肺炎放线杆菌6种主要毒力因子基因:apxⅠ、apxⅡ、apxⅢ、apxⅣ、apfa和omp,以重组蛋白rApxⅠ、rApxⅡ、rApxⅢ和rOMP组合免疫小鼠作为试验I组,重组蛋白rApxⅠ、rApxⅡ、rApxⅢr、OMPr、ApxⅣ和rApfa组合免疫小鼠作为试验Ⅱ组,PBS为对照组,分3次免疫小鼠,采用背部皮下多点注射,每次间隔2周,免疫剂量为0.2 mL/只,3免后1周分别以APP1型菌Shope 4074株(5×109cfu)和APP2型菌S1536株(5×1010cfu)进行攻毒试验。通过小鼠保护率与抗体效价的相关性研究、肺部病理变化及肺脏细菌的分布情况等指标进行综合评价。结果显示,试验Ⅰ组4种重组蛋白特异性抗体水平显著高于其他两组(P<0.05),对APP1型菌攻毒的保护率(9/10)明显高于试验Ⅱ组(5/10)和对照组(0/8),小鼠的免疫保护率与抗体效价之间存在显著正相关;且该组对APP2型菌攻毒的保护作用(无肺脏损伤)也明显优于其它两组(典型肺部损伤)。间接免疫荧光试验表明试验Ⅰ组对肺脏细菌的清除效果也明显优于其他两组。本试验揭示试验Ⅰ组对不同血清型APP攻击能够提供很好的交叉保护作用,从而为猪传染性胸膜肺炎新型疫苗的研制提供参考。     

鸽源冠状病毒PSH株纤突蛋白基因的遗传变异分析

对鸽源冠状病毒PSH株纤突蛋白基因进行了RT—PCR扩增、克隆和测序。结果表明，PSH株S基因由3504个核苷酸组成，编码1条由1167个氨基酸残基组成的多肽。S基因编码产物裂解后形成的S1和S2亚单位分别由541和626个氨基酸残基组成。PSH株S蛋白切割识别位点为精氨酸-精氨酸-苯丙氨酸-精氨酸-精氨酸（RRFRR），与多数鸡传染性支气管炎病毒（IBV）切割识别位点相似（RRF／SRR）。与GenBank中其他冠状病毒毒株S基因的推导氨基酸序列相比较，PSH株与IBV参考毒株的同源性为79．3％～99．6％，而与其他冠状病毒包括火鸡蓝冠病病毒和SARS病毒的同源性均小于37．8％。表明，鸽源冠状病毒PSH株属于第3抗原群冠状病毒，且与IBV亲缘关系较近。     

猪IL-18基因的原核表达及重组蛋白的纯化

以pcDNA3．1-pIL-18为模板，采用PCR技术扩增到了猪白细胞介素18（IL-18）的成熟蛋白基因，通过KpnⅠ＋SacⅠ双酶切及连接反应，构建了pET32c—pIL—18原核表达质粒。经过限制性内切酶分析、PCR鉴定及DNA序列测定证实，重组质粒中的基因片段连接正确。之后，重组质粒转化大肠杆菌BL21（DE3），于37℃、1．0mmol／L IPTG条件下诱导表达。菌体裂解产物经SDS—PAGE分析，在分子质量约为33ku处出现了预期的目的蛋白。用8mol／L脲对表达产物变性，经Ni^2＋NTA柱纯化，透析复性，得到了纯化的IL-18蛋白。Western—blot分析证实，纯化的重组IL-18蛋白具有反应活性。上述研究结果为重组IL-18的应用奠定了基础。     

Isolation and Identification of a Variant Porcine Epidemic Diarrhea Virus and the Immune Efficacy of Its Inactivated Vaccine

This study was aimed to isolate a mutant strain of porcine epidemic diarrhea virus and prepare a PEDV inactivated vaccine with high valence by suspension culture process for immunizing against PEDV effectively in China.200 small intestines and theirs contents of diarrhea piglets died of diarrhea,collected from many large-scale pig farms in China,were detected by RT-PCR and sequenced,a mutant strain of porcine epidemic diarrhea virus was selected and put on the suspension-cultured Vero cells in a 2 L reactor for virus isolation and continuous cell culture,the harvested virus suspension,which was identified and determined TCID₅₀,was inactivated by formaldehyde and mixed with aluminum hydroxide gel adjuvant to prepare PEDV inactivated vaccine.After its physical behavior,stability viscosity,sterility test were checked out,the safety and immune efficacy were studied by immunizing the pregnant pigs and theirs piglets.The results were as follows:86 samples were detected positive in 200 samples,cytopathy occurred after the mutant strain samples screened were passaged to 5th generation,the virus suspension was harvested in 10th generation and identified as a mutant strain of PEDV,named PEDV-GF10 strain.The virus titer of harvested virus suspension was measured up to 1×10^8.0 TCID₅₀/mL after concentrated.After the vaccine was checked out,the sows,40 and 25 days before delivery in experimental groups,were injected into Xuehai acupoint with 4 mL vaccine and the pigs in blank group were free of immunifications.The results showed that there were no obvious differences in the production status of the sows in experimental groups and blank group and the temperature of theirs 3-day-old healthy piglets injected different doses of vaccine,and the vaccine was safe to the sows and piglets.Forty 3-day-old piglets producted by pregnant sows in experimental groups and blank group were randomly selected and taken 4 mL 1×10^8.0TCID₅₀/mL F10 virus culture.The PEDV morbidity of piglets in blank group was 100% after injection and the antibodies were negative;10% piglets in blank group had mild diarrhea symptoms,the protection rate was up to 90%,antibody of passive immunity in piglets lasted for more than 35 days.Virus titer of mutant strain of PEDV-GF10 improved a lot by suspension cell culture,the PEDV-GF10 inactivated vaccine was safe,and could effectively prevent and control the variation strain of PEDV in China.     

Research Progress of Canine Coronavirus and Feline Coronavirus

Canine coronavirus (CCoV) and feline coronavirus (FCoV) belong to α-genus coronavirus of coronavirus family,porcine transmissible gastroenteritis virus (TGEV),porcine epidemic diarrhea virus (PEDV) also belong to the same genus.Genetic evolution analysis showed that different genotype of the virus could produce new variant strains through gene recombination,which caused great obstacles to the diagnosis and control of the disease.β-genus coronaviruses include bovine coronavirus (BCoV),canine respiratory coronavirus (CRCoV) and severe acute respiratory syndrome coronavirus (SARS-CoV).Among them,CRCoV has the highest homology with BCoV,but there are great differences in genomic structure,pathogenic mechanism and infection symptoms between this kind of coronavirus and α-coronavirus.CCoV and FCoV are widely spreading around the world,characterized by high morbidity and low mortality.Due to the characteristics of RNA virus and the influence of environmental selection pressure,the viruses continue to mutate and evolve,and new virulent strains appear one after another.The virulence of feline infectious peritonitis virus (FIPV) is greatly enhanced,some specific point mutations in the virus genome change the cellular tropism against the host.The pathogenesis of the virus mainly depends on the antibody-dependent enhancement (ADE) induced by virus infection.The epidemiological investigation and prevention and control of CCoV and FCoV should not only rely on the single factor of vaccine immunity,but also comprehensively consider the virulence of the virus,environmental conditions,pet self-immune resistance, and so on.The identification of CCoV and FCoV should be based on clinical symptoms,combined with routine hematological examination,serum biochemical examination and laboratory diagnosis techniques to prevent false positive and false negative results.     

1株高致病性血凝性脑脊髓炎病毒的分离与鉴定

利用细胞培养方法从临床表现神经症状的死亡仔猪脑组织内分离获得1株病毒,同时对该病毒进行电镜负染观察、昆明种小鼠致病性试验、RT-PCR检测和部分基因测序分析。电镜观察可见细胞培养物中有大量的直径约115 nm的冠状病毒样粒子;接种病毒的小鼠均出现典型的神经症状,死亡率100%;血凝性脑脊髓炎病毒(HEV)特异性引物RT-PCR扩增结果阳性;其S基因序列与GenBank中登录的HEV-67 N相应基因序列的同源性高达99.6%。结果表明,分离的病毒为猪血凝性脑脊髓炎病毒,分离株暂命名为HEV-CC-2007。     

小鼠4、8-细胞胚胎单卵裂球体外培养体系的研究

以CZB为基础培养液,培养小鼠4、8-细胞胚胎单卵裂球,研究葡萄糖、牛磺酸和猪输卵管上皮细胞共培养在其体外发育中的作用。结果表明:牛磺酸添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为29%、30%和14%、14%,无显著性差异(P>0.05)。添加葡萄糖后,4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%和19%,有显著提高(P<0.05)。含有葡萄糖而牛磺酸的添加与否对4、8-细胞胚胎单卵裂球的囊胚发育率分别为36%、38%和19%、20%,无显著性差异(P>0.05)。各组内4、8-细胞胚胎单卵裂球形成的囊胚细胞数分别为(10.44±1.24～(12.43±1.18)和(7.57±0.97)～(8.48±1.16),均无显著性差异(P>0.05)。4、8-细胞胚胎单卵裂球与猪输卵管上皮细胞共培养,其囊胚发育率分别为47%和26%,囊胚细胞数分别为(17.57±1.13)和(11.43±0.92),均高于单一培养(P<0.05)。     

大熊猫犬冠状病毒部分S基因的克隆与全S基因序列分析

从重庆某动物园死亡的大熊猫肝脏中分离到一株病毒，经鉴定为犬冠状病毒（命名为CCV DXMV）。本实验根据CCV K378和CCV Insavc-1株基因序列设计合成了普通PCR引物SF1-SR1和SF2-SR2，以及半套式引物SF3-SR3、SR13、SF13、SF4-SR4和SF14，分段扩增了CCVDXMV株部分S基因，得到了368bp、792bp、737bp、1178bp和985bp5个基因片段。将纯化的RCR产物分别克隆到pGEM—T载体中，通过筛选和鉴定，获得了5个阳性重组质粒。将重组质粒送生物公司测序，将测得的基因序列与先前测定的该株病毒部分S基因序列，拼接成全S基因序列，并与其它株CCV及TGEV HN2002和FIPV79—1146株的S基因序列进行分析比较，绘制进化树。结果表明，该株病毒与CCV K378株的同源性最高，达到99．4％；而与CCV 23／03株的同源性最低，为56．9％；与FIPV和TGEV分别有90．4％和82．1％的同源性。