表达禽流感病毒核蛋白基因重组禽痘病毒的构建及其免疫保护性研究

 从含有禽流感病毒A/Goose/Guangdong/1/96 (H5N1)核蛋白 (NP)基因的质粒pUCNP中切下NP基因片段 ,将其亚克隆到pSY5 38质粒 ,再将带有痘苗病毒启动子P11的LacZ基因也平端克隆到pSY5 38质粒 ,然后切下同时含有NP及LacZ基因的片段 ,再亚克隆到禽痘病毒载体pSY6 81,从而构建出表达核蛋白基因的重组禽痘病毒转移载体pSY(NP +LacZ)。应用脂质体介导的方法将转移载体转染已感染禽痘病毒S FPV 0 17的鸡胚成纤维细胞 ,在X gal存在的条件下 ,利用蓝白斑筛选、数轮蚀斑纯化以及PCR、Western blot鉴定 ,结果证明 ,获得了能高效表达AIVNP蛋白的重组禽痘病毒rFPV NP。SPF鸡体内的免疫保护试验结果表明 ,它能够诱导机体产生较高水平的NP特异性抗体 ,并对H5N1和H7N1这 2种亚型高致病力禽流感病毒的攻击提供一定的保护。     

昆明系和Balb／C系小鼠用于显性致死试验的比较研究

本研究选用昆明系和Ｂａｌｂ／Ｃ系小鼠同时进行了显性致死试验的对比试验。试验一选用封闭群昆明系９￣１０周龄的雄性小鼠７８只，随机分成６组，其中５个剂量组分别灌服１６０、５３、１６ｍｇ／ｋｇ体重的复方硝基酚钠和１５、３０ｍｇ／ｋｇ体重的环磷酰胺，另一组为空白对照组。受试物组每天给药一次，连续５ｄ后，按１：２的雄雌性比例与正常的９￣１０周龄封闭群昆明系雌鼠进行交配，每周交配一批，连续交配８批。试验二除选     

Prokaryotic Expression of BPIV3 NP Protein and Its Immunoenhancing Effect on the BPIV3 Inactivated Vaccine

【Objective】 This study was aimed to verify whether the NP protein of Bovine parainfluenza virus type 3 (BPIV3) could enhance the immune effect of BPIV3 inactivated vaccine【Method】 The antigenicity of the protein encoded by NP gene was analyzed by bioinformatics softwares,and the antigenic region was screened.The truncated NP gene sequence of BPIV3 was amplified by PCR and connected to pET-32a(+) plasmid.Then the high-purity BPIV3 NP protein was obtained by E.coli prokaryotic expression system and Ni affinity chromatography.It was confirmed by Western blotting.BPIV3 was inactivated with 0.3% formaldehyde and mixed with Freund's adjuvant 1:1 to prepare inactivated vaccine.Eight New Zealand White rabbits were randomly divided into four groups with two rabbits in each group,including inactivated vaccine group,NP protein group,inactivated vaccine and NP protein mixed group and control group.Blood samples were collected before and every 7 days after immunization.The levels of specific antibodies and neutralizing antibodies in New Zealand White rabbits of the four groups were measured and compared by indirect ELISA and virus neutralization test.【Result】 DNAStar analysis showed that the average antigen index of amino acid region 193-368 of NP protein was 0.4-1.7,and the hydrophilic index was 0-1.5,which proved that this region had strong antigenicity and hydrophilicity.The NP gene was amplified by PCR and the recombinant expression vector was constructed.Gene sequencing showed that the recombinant expression vector was consistent with the expected results.The results of SDS-PAGE showed that NP protein was highly expressed with a molecular weight of 50 ku and expressed in the form of inclusion body.Western blotting showed that the expressed protein had strong reactivity.The results of ELISA showed that 28 days after immunization,the specific antibody titer of the control group was 0,and the specific antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group reached 1:2¹¹,1:2¹⁷ and 1:2¹⁸,respectively.The results of virus neutralization test showed that 28 days after immunization,the neutralizing antibody titer of the control group was 0,and the neutralizing antibody titers of inactivated vaccine group,NP protein group and inactivated vaccine and NP protein mixed group were 1:2^3.32,1:2^4.48 and 1:2^4.98,respectively.【Conclusion】 BPIV3 NP protein could enhance the immune effect of BPIV3 inactivated vaccine.Adding NP protein to the inactivated vaccine could be used as a new vaccination method of BPIV3 inactivated vaccine.     

杂交鹅掌楸转双价抗病基因影响因子研究

本研究利用在固体培养基上长期保存的杂交鹅掌楸胚性愈伤为外植体,对杂交鹅掌楸进行了遗传转化的研究.对于影响转化效果的众多影响因素,本研究主要讨论了乙酰丁香酮、菌液浓度、侵染时间对杂交鹅掌楸转化结果的影响.试验结果表明,在预培养阶段,乙酰丁香酮的使用有利于转化效率的提高,最佳条件时浓度100 mg/L,预培养4 d.而在共培养阶段,乙酰丁香酮的使用和延长共培养时间并不能显著提高杂交鹅掌楸的遗传转化效率.同时,在侵染时,不同菌液浓度和不同侵染时间对转化结果的影响也无显著的差异.通过PCR检测经过筛选培养获得的阳性植株,初步验证了外源基因已经整合到杂交鹅掌楸基因组中.     

马尔堡病毒核蛋白的原核表达及纯化

目的以原核表达的方式获得了马尔堡病毒的核蛋白,并纯化。方法将马尔堡病毒核蛋白基因亚克隆到原核表达质粒pET-28(a)中,构建重组表达质粒pET-28(a)-M-NP,并将重组质粒用热休克方法转化BL21(DE3)感受态细菌,用IPTG诱导蛋白表达后,以SDS-PAGE分析表达形式及表达量,并利用His-Band Ni+柱纯化。结果成功构建重组表达质粒pET-28(a)-M-NP,以1.0mmol/L终浓度的IPTG在37℃诱导表达5h后,破菌沉淀中有目的蛋白表达。结论成功表达并纯化了马尔堡病毒的核蛋白,为后续研究奠定了基础。     

长期氮磷化肥配施对不同种植体系土壤水分的影响

利用长期定位实验,在黄土高原旱地研究了22a氮磷化肥配施下不同种植体系土壤水分的变化状况,为合理利用水肥资源提供参考。在小麦连作、豌豆-小麦（2a）＋糜子轮作、红豆草-小麦（2a）轮作、豌豆-小麦（2a）＋玉米轮作、玉米-小麦（2a）＋糜子轮作、小麦（2a）＋糜子-玉米轮作6个不同种植体系中,测定土壤剖面（0-300cm）含水量、储水量。结果表明：不同种植体系土壤含水量在土壤剖面上的分布不同,所有处理在土壤剖面140-300cm土层含水量均低于0-140cm土层含水量。不同种植体系在土壤剖面不同土层的储水量不同,在0-300cm土壤储水量各处理表现为豌豆-小麦（2a）＋玉米轮作（644.8mm）〉小麦（2a）＋糜子-玉米轮作（599.6mm）〉豌豆-小麦（2a）＋糜子轮作（582.3mm）〉红豆草-小麦（2a）轮作（574.0mm）〉玉米-小麦（2a）＋糜子轮作（550.8mm）〉小麦连作（536.9mm）。轮作体系0-300cm土壤储水量均高于小麦连作。粮豆轮作土壤储水量高于小麦连作,是黄土高原地区较好的种植方式。     

猪博卡病毒NP1基因的克隆及其生物信息学分析

根据Gen Bank发表的猪博卡病毒主要抗原基因NP1基因设计1对引物,采用降落PCR方法进行扩增,克隆后测序,并对该基因进行生物信息学分析。结果表明:成功克隆到猪博卡病毒NP1基因;NP1蛋白的二级结构中自由卷曲含量最高(51.77%),无跨膜蛋白,含有1个糖基化位点、30个磷酸化位点,有6个线性B细胞优势抗原表位,分别位于NP1蛋白的7—14位、27—50位、59—73位、85—94位、102—108位、151—155位。     

黑龙江农田土壤壬基酚及其短链聚氧乙烯醚残留调查

对黑龙江土壤样品中的壬基酚(NP)、壬基酚一氧乙烯醚(NP1EO)和壬基酚二氧乙烯醚(NP2EO)的残留状况开展研究。结果表明,NP、NP1EO和NP2EO均有不同程度的检出,检出率在68.46%~95.38%之间;NP、NP1EO和NP2EO的浓度分别为ND~156.8μg kg-1、ND~15.70μg kg-1和ND~13.63μg kg-1,均值分别为22.02μg kg-1、4.04μg kg-1和2.15μg kg-1;进一步分析,已检出的样品中,NP在0~10μg kg-1的比重最大,NP1EO在4~6μg kg-1比重最大,NP2EO在1~2μg kg-1比重最大。NP的残留水平高于NP1EO和NP2EO,说明NP相对较难降解,更易在土壤中累积。     

差分方程2~NP序列周期解顺序

一维单峰映象差分方程在种群生态学和其他某些学科中有着较重要的地位。本文简述了它的迭代过程的几个特征,给出了倍周期分岔2NP序列解的顺序表达式,并与X_(n+1)=1-μX_n~3和X_(n+1)=μX_n(1-X_n)/(1-X_nβ)两种差分方程中的顺序作了对比。