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.

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.

Objective:To investigate the risk factors of hepatorenal syndrome (HRS) in patients with Hepatitis B-related acute-on-chronic liver failure(ACLF).Methods:736 Hepatitis B-related ACLF patients were enrolled and divided into HRS group and non-HRS group.The baseline demographic,clinical,biochemical,virological features,the bulk of liver,and whether complications occured or not in these patients were collected.Univariate analysis and Logistic regression were applied to identify the influencing factors of HRS.The survival curve was established to analyze the association between HRS and 3-month mortality of ACLF.Results:The ratio of hepatitis B virus e antigen (HBeAg),the data of cholesterol (CHOL),alpha-fetoprotein (AFP),prothrombin activity (PTA),left hepatic size,and right hepatic size in HRS group were lower than non-HRS group.While the age,the data of serum cretinine (Cr),and plasma ratio of international standardization (INR) in HRS group were higher than non-HRS group,the difference were statistically significant (P ＜ 0.05).The ratio of the hepatic encephalopathy(HE),primary peritonitis,mycotic infection,upper gastrointestinal hemorrhage,prothrombin activity (PTA),and right hepatic size were the risk factors of HRS in patients with Hepatitis B-related ACLF.HRS was associated with increased 3-month mortality in Hepatitis B-related ACLF patients.Conclusion:The ratio of the hepatic encephalopathy (HE),primary peritonitis,mycotic infection,upper gastrointestinal hemorrhage,prothrombin activity (PTA),and right hepatic size were the risk factors of HRS in patients with Hepatitis Brelated ACLF.It suggests that we should prevent this complication actively and intervene it as early as possible.

目的 探讨肿瘤坏死因子(TNF-α)基因多态性与乙型肝炎病毒慢性感染结果之间的关系以及乙型肝炎病毒慢性感染患者血清中TNF-α水平在慢性乙肝发展中的临床意义.方法 运用聚合酶链反应-限制性片段长度多态性分析的方法检测TNF-α基因启动子区-238位点单个核苷酸多态性在不同临床类型的慢性HBV感染者及健康对照者中的分布频率;应用ELISA方法检测血清TNF-a浓度水平.结果 TNF-α-238位点G/A和G/G基因型频率以及A等位基因频率分布在实验组和健康对照组的差异无统计学意义.慢性乙型肝炎组、肝硬化组、乙型肝炎肝衰竭组和健康对照组比较,血清TNF-α均有不同程度升高,TNF-a (ng/L)取对数值后经方差分析,差异有统计学意义(P＜0.01);组间两两比较,差异均有统计学意义(P＜0.01),血清TNF-α水平乙型肝炎肝衰竭组＞肝硬化组＞慢性乙型肝炎组＞健康对照组.结论 TNF-α-238位点基因型与乙型肝炎易感性无明显相关性.TNF-α与乙型肝炎类型、肝损伤程度有密切关系.

AIM:To investigate the clinicopathologic features of chronic active Epstein-Barr virus hepatitis (CAEBVH) as well as its diagnosis,differential diagnosis,treatment and prognosis.METHODS:We presented the clinical manifestations,histopathological characteristics,diagnosis,treatment and prognosis of two cases of CAEBVH.A literature review was also performed to summarize the characteristics of this clinical entity.RESULTS:Of two young male patients,one presented with intermittent fever,jaundice,hepatosplenomegaly and abnormal liver function,the other had abnormal liver biochemical tests and symptoms including edema of lower limbs,yellowish urine,fatigue,splenomegaly,and central nervous system symptoms.Histopathologic examination of liver biopsies revealed varying degrees of macrovesicular steatosis and fibrosis,scattered lobular necrosis,beaded sinusoidal lymphocytic infiltration,portal inflammation and interface activity.Electron microscopic investigation showed chronic hepatitis along with steatosis of liver cells and fibrosis.No mylinoid body,special lysosome,glycogen storage or hepatitis B surface antigens were found.In situ hybridization (ISH) for EBV early RNA (EBER)showed EBER-positive nuclei of lymphocytes.Both patients ended in death.The course was 2years and 4 months for case 1 and 13 years and 5mo for case 2.CONCLUSION:CAEBVH has no specific clinical features,which makes it easy to reach a misdiagnosis.Pathologic features include macrovesicular steatosis,fibrosis,beaded sinusoidal lymphocytes infiltration,scattered lobular necrosis,interface activity and portal inflammation.EBER-positive nuclei of lymphocytes can be detected by ISH.This disease has a poor prognosis and early diagnosis is pivotal for appropriate clinical management.