禽呼肠孤病毒分子生物学研究进展

本文就十几年来有关呼肠孤病毒的特性、基因组、基因重组、病毒蛋白、诊断方法等分子生物学的研究进行了综述,并指出了禽呼肠孤病毒分子生物学研究中存在的问题,及进一步研究的方向。     

减毒沙门氏菌为载体传递DNA疫苗诱导对新城疫病毒的免疫保护

探讨了以减毒鼠伤寒沙门氏茵为栽体传递新城疫病毒DNA疫苗的安全性、免疫原性和可行性。将含新城疫病毒(NDV)F48E9株融合蛋白(F)基因的真核表达质粒pcDNA3-F的重组减毒鼠伤寒沙门氏菌ZJ111株(ZJ111／pcD-NA3一F菌株)，以10^8CFU进行首免，2周后二免，三免后4周攻击强毒株F48E9，观察其安全性和免疫原性，同时设只含空载体pcDNA3的ZJ111／pcDNA3菌株对照及口服PBS对照。结果表明：重组ZJ111／pcDNA3-F菌株具有良好的安全性。对强毒株攻击的保护率达64．7％。重组ZJ111／pcDNA3-F菌株不仅能诱导雏鸡产生NDVELISA抗体，而且诱导产生的法氏囊B淋巴细胞和胸腺T淋巴细胞增殖反应显著高于ZJ111／pcDNA3时照组。这些结果提示，减毒沙门氏菌为载体不仅可直接将NDVF基因呈递给鸡体细胞进行表达，产生抗NDV的体液免疫，而且还可诱导细胞免疫应答。     

Feline vaccine‐associated sarcomagenesis: Is there an inflammation‐independent role for aluminium?

Aluminium has been found in feline vaccine‐associated sarcomas. In this study, we investigated the potential for aluminium to contribute directly to tumourigenesis. Our results indicated that an aluminium hydroxide adjuvant preparation was cytotoxic and mutagenic in human‐Chinese hamster ovary (CHO) hybrid cells in vitro. Moreover, CHO cells deficient in DNA double strand break (DSB), but not single‐strand break (SSB), repair, were particularly sensitive to aluminium exposure compared with repair proficient cells, suggesting that aluminium is associated with DSBs. In contrast to CHO cells, primary feline skin fibroblasts were resistant to the cytotoxic effects of aluminium compounds and exposure to an aluminium chloride salt promoted cell growth and cell cycle progression at concentrations much less than those measured in particular feline rabies vaccines. These findings suggest that aluminium exposure may contribute, theoretically, to both initiation and promotion of tumours in the absence of an inflammatory response.     

表达HAV结构基因的重组腺病毒鉴定及细胞病变分析

利用RT-PCR方法,从HAV-L8株的RNA中扩增出结构基因(vp3 vp1),克隆到穿梭质粒pXCX2NotI上。采用磷酸钙-DNA共沉淀技术,将腺病毒载体pJM17与pXCX2-CMV-HAV共转染293细胞。在光学显微镜下观察到明显的细胞病变(CPE);经RT-PCR、免疫荧光染色和蛋白印迹鉴定证明HAV的结构基因已插入到腺病毒基因组中;在电子显微镜下观察发现有二十面体的病毒颗粒。以上结果表明获得了重组腺病毒rAdHAV,纯化后的rAdHAV滴度为1×109.0TCID50/mL。     

抗原表位及其在血吸虫疫苗研究中的应用

血吸虫病是世界上危害最严重的寄生虫病之一，其疫苗的研究是世界血吸虫病防治的一项工作重点也是一大难题。抗原表位是疫苗设计和免疫识别的基础，本文对抗原表位在疫苗设计中的分子理论基础做了阐述．介绍了其在血吸虫疫苗设计中的应用，并提出了一些问题与展望。     

致病性大肠杆菌苗用菌株的原生质体融合的研究

对筛选的5株大肠杆菌(E2:O2、E6:O78、E7:O8、E9:O9、E22:O138)进行药敏试验以及致病性大肠杆菌原生质的制备、融合和筛选试验。结果表明:①5株大肠杆菌对多种抗菌素存在抗药性,并且这种抗药性能进行培育,同时,在原生质的融合过程中能递进;②大肠杆菌原生质体选择的最佳组合是:溶菌酶2.0 g/L,处理时间是20 m in;③大肠杆菌的融合率在10-8左右,大肠杆菌之间的融合率为10-6左右;共选出3株融合菌株为E2-E6、E6-E7和E2-E22,各融合株的生化特征介于起源菌株的生化特征之间,有2株融合株的抗原特征含原菌株各自的抗原成分。     

高邮鸭和金定鸭胸肌早期发育及MyoD1基因表达分析

 
采用qPCR分析生肌调节因子MyoD1基因在高邮鸭和金定鸭胚胎期及初生早期(13、17、21、25、27胚龄和出雏后7日龄)胸肌发育中的表达模式以及与胚胎和胸肌发育的相关性。结果表明,MyoD1 mRNA在两个品种鸭胸肌早期发育中表现出一致的表达规律,均呈“波浪形”,在13胚龄时表达量相对较高,17胚龄时下降,21胚龄时上升到最高,随后下降,出雏后又维持较高水平;品种间比较结果显示除了在25胚龄时金定鸭胸肌中MyoD1 mRNA表达量稍低于高邮鸭,在其他所检测的胚龄/日龄中,金定鸭胸肌中MyoD1 mRNA表达量均高于高邮鸭胸肌中的表达量(P>0.05);高邮鸭胸肌MyoD1 mRNA表达与胚胎和胸肌发育无显著相关性,而金定鸭胸肌中MyoD1 mRNA的表达与其胚胎和胸肌发育呈强负相关(P胚重=0.048;P胸肌重=0.006)。MyoD1基因参与鸭胚胎期及出雏早期胸肌的发育,胸肌中MyoD1基因表达分析研究为进一步深入研究MyoD1基因在胚胎期胸肌发生过程及其调控机理中的功能提供一定的理论依据     

禽腺联病毒全基因组的克隆及感染性病毒的拯救

为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序。序列分析表明,AAAV YZ-1株的基因组为4684bp,两端具有141bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%。将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建。     

Study on Synergistic Effect of VA5 Immunopotentiator on Pigeon NDV Inactivated Vaccine

This study was designed to evaluate the synergistic effect of VA5 immunopotentiator on pigeon ND4416 inactivated vaccine.160 healthy pigeons were randomly divided into four groups,including ND4416 strain inactivated vaccine group (NDV group),ND4416 strain inactivated vaccine and immunopotentiator (VA5) mixed group (NDV+VA5 group),La Sota inactivated vaccine group (La Sota group) and normal saline as the control group (C group),to assess their vaccine efficacy against virulent pigeon NDV by serological analysis and animal testing.Pigeons sera were collected at different time points after immunization and measured the HI antibody titer of each group.The results showed that VA5 immunopotentiator significantly improved the serum antibody level of pigeons immunized with pigeon Newcastle disease inactivated vaccine (P<0.05).In addition,comparative test of spleen lymphocyte transformation was conducted at various time points after immunization.The results indicated that VA5 effectively stimulated the lymphocyte transformation of immune pigeon.Pigeons in each groups were challenged with ND4416 strain at the 30th,90th and 180th d after immunization.The results presented that the NDV+VA5 group had 100% protection rate and higher than La Sota group.The duration of immunization test showed that the antibody titer of NDV+VA5 group reached peak 11.20log₂ at the 21st d,remained 7.50log₂ at 180th d,and the protection rate remained 100% at 180th d.It indicated that VA5 immunopotentiator sustained the immune duration of pigeon NDV vaccine up to 180 d.Moreover,the in vitro detoxification test results suggested that VA5 immunopotentiator reduced the in vitro detoxification cycle of pigeons after challenge.Overall,this study suggested VA5 immunopotentiator could significantly improve the immune efficacy of pigeon Newcastle disease inactivated vaccine,which provided a basis for further enhancing the efficacy of Newcastle disease vaccine,as well increased experimental data for the application of immunopotentiators.     

鸡抗鸭病毒性肝炎高免卵黄抗体的研制及应用

应用雏鸭病毒性肝炎病毒（DHV）弱毒疫苗和DHV油乳剂灭活苗多次免疫产蛋鸡，制备鸡抗鸭病毒性肝炎高免卵黄抗体，自制鸭病毒性肝炎病毒高免卵黄抗体的疗效取决于卵黄抗体的中和效价及治疗的时间。实验室试验及临床应用结果表明，卵黄抗体的中和效价应达2^8以上，并且在感染DHV 24h以内对雏鸭进行肌肉注射，治疗效果最为理想。