【作者】 郜玉钢；

【导师】 王树志；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2005， 博士

【摘要】 1 从吉林省长春市双阳区梅花鹿流产胎儿肝脏病料中分离出的病毒,接种于MDBK传代细胞后出现了BVDV典型而规律的细胞病变,其理化特性与BVDV相同,致细胞病变作用可被BVDV国际标准株C₂₄V株的牛阳性血清所阻断,电镜负染观察病料接种MDBK细胞的F₁代浓缩病毒液,可见典型的BVDV粒子形态,从F₁代浓缩病毒液中分别扩增出402bp(NS_2-3)和706bp（E₀）的目的片段,证明该毒株是BVDV,命名为CCSYD株。 2 对从吉林不同地区分离的4株（CCSYD株,CCJYD株、CCKCD株、JLCYD株）BVDV的NS_2-3基因外源序列插入区进行了RT-PCR扩增、克隆和测序,并与BVDV其他株进行了同源性分析,CCSYD株属基因Ⅰb亚型,CCJYD株、CCKCD株、JLCYD株属待定基因型。 3 将CCSYD株BVDV的E₀基因RT-PCR扩增目的片段进行了克隆和测序,将其与已报道的瘟病毒代表株相应序列做了比较,预测了E₀蛋白的抗原表位、亲水性和等电点等。以E₀基因为判定依据,CCSYD分离株也为基因Ⅰb亚型,E₀基因的核苷酸序列可做为BVDV基因分型的依据。 4 成功构建了原核表达质粒pET28a/E₀,重组菌在IPTG诱导下能表达目的蛋白,其含量占菌体总蛋白的9.25%。 5 成功构建了真核表达质粒PAX1/E₀,并用脂质体转染BHK-21细胞,RT-PCR法检测到E₀目的基因在BHK-21细胞进行了转录,间接ELISA法检测到已表达目的蛋白。 6 用梅花鹿源BVDV基因苗（PVAX1/E₀）不同免疫剂量和免疫次数免疫家兔,既可产生体液免疫又可产生细胞免疫应答。基因苗高剂量组比低剂量组体液免疫应答水平和细胞免疫应答水平高,基因苗免疫次数对体液免疫应答水平和细胞免疫应答均无影响。免疫的第42天基因苗免疫组抗体水平达到高峰,基因苗免疫组（免疫剂量1mg/ml以上）BVDV抗体应答水平高于CCSYD灭活苗和C₂₄V灭活苗免疫组,但免疫家兔的28天以前的结果则相反;免疫家兔产生的细胞免疫应答水平基因苗组均低于CCSYD灭活苗和C₂₄V灭活苗免疫组。 7 采用组织细胞培养方法,测试了利巴韦林、黄芪、鱼腥草、干扰素a2b、莪术油、双黄连粉针剂在MDBK细胞体外培养中的最大安全浓度（最低稀释度）分别为(2¹⁴)、（2⁵）、（2⁸）、（2⁵）、（2⁷）、（2⁷）。药物抗梅花鹿源BVDV作用由强到弱的顺序:莪术油、鱼腥草、黄芪、干扰素、利巴韦林、双黄连。更多还原

【Abstract】 Bovine viral diarrhea virus （CCSYD strain） of sika was isolated and identified in this study. BVDV NS_2-3 genes of CCSYD strain and three isolated BVDV strains （CCTYD, CCKCD and JLCYD） were cloned and sequenced and E₀ gene of isolated CCSYD strain was cloned, sequenced and expressed on eukaryote and prokaryote. The immunogenicity of isolated CCSYD genetic vaccine was tested. Medicine sensitivity of CCSYD isolated from BVDV was selected. The results were shown as follows:1 The virus isolated from liver of an aborted fetus of sika in Shuangyang district of Changchun in Jilin province was identified systematically. The results showed that when the virus was inoculated to MDBK cell line, presented itself classical and regular CPE of BVDV and its physicochemical assays is the same as BVDV. CPE can be blocked by positive serum of BVDV international standard C₂₄V strain. Classical BVDV particles were observed by negative staining electron microscope assays in F₁ compressed viral fluid inoculated by MDBK cell. 402bp(NS_2-3) and 706bp（E₀） of F₁ viral fluid was amplified by RT-PCR. All the above results proved this virus was BVDV and was designated CCSYD.2 Exogenous sequence insertion domain of NS_2-3 of CCSYD, CCTYD, CCKCD and JLCYD isolated from different districts of Jilin province was amplified by using RT-PCR. The fragment was transformed into JM-109 after connecting to pMD18-T, thus, recombinated cloning plasmid pMD18-T/NS_2-3 was established and sequenced, tested for nucleus acid sequence, therefore, amino acid sequence was inferred and compared. The results showed homeology of nucleotide sequence of CCSYD NS2-3 compared with those of other strains were VEDEVAC 100%, 184 99.3%, H 96.6%, ZM95 98%, OSLOSS 94.9%, Draper 92.6%, ILLC 92.4%, SNC 91.1%, YAK 82.3%, D 83.0%, 3142 84.8%, 3387 84.3%, C₂₄V 83.2%, NADL 83.4%, SD1 83%, Singer 82.8%, 06-APR-1993 77.0%, NY-1 75.8%, CCKCD 42.3%, CCJYD 42.3%, JLCYD 42.5%, respectively. Compared with other strains, the nucleotide sequence homeology of NS_2-3 of CCSYD were VEDEVAC 100 %, 184 99.4%, H 94.5%, ZM95 95.7%, OSLOSS 93.3%, Draper 93.9%, ILLC 93.3%,SNC 92.0%, YAK 88.3 %, D 90.2%, 3142 92.0%,3887 89.0 %, C₂₄V 86.5%, NADL 88.3 %, SD1 90.8%, Singer 87.1%, 06-apr-1993 82.8% NY-1 72.4%, CCKCD 28.2%, CCJYD 28.2%, JLCYD 28.2%, respectively. The CCSYD strain has no exogenous sequence insertion, gene recombination, gene rearrangement and gene deficiency, however, some nucleotide sequences and aminoacid sequences were replaced. The other three strains existed exogenous sequence insertion and gene rearrangement. The results indicated that BVDV CPE not only related to exogenous sequence insertion, gene recombination, gene arrangement, gene deficiency but also related to nucleotide replacement. CCSYD gene belongs to subtype Ib. CCJYD, CCKCD, JLYD belong to some unknown gene types.3 Eo gene of CCSYD strain isolated from the liver of an aborted fetus of sika was amplified utilizing RT-PCR. The fragment was transformed into JM-109 after connecting to pMD18-T, thus, recombinated cloning plasmid pMD18-T/ Eowas established and sequenced. The antigen epiposition, hydrophilicity and the isoelectric point of Eo were predicted by comparetion with the pestvirus sequences reported previously. The results showed that the CCSYD isolated strain was 681bp, which compared with 9 strains BVDV （VEDEVAC, Bega, C24V ILLC, NADL, OSLOSS,R1935, SD-1,Y546）, 7 strains hog choler virus （ALD, Brescia, C, GPE, JL, LN9912,SM） and 3 strains border disease virus （BD31, C413, BDVX 818） the homeology of nucleotides sequences were 98.6%-84.8%, 76.1%-74.7%, 77.0%-76.7%, respectively; The homeology of amino acid sequences were 98.7%-91.6%, 79.9%-78.3%, 83.9%-80.6%, respectively. The isoelactric points of Eo protein of CCSYD were 7.61, the electronic charge was 1.99 at pH=7. The higher domain of antigenicity exponent and hydrophilicity peak value were 8-16, 23-29, 59-67, 70-81, 96-109, 114-122, 127-134, 137-143, 165-172, 186-198, 213-221. In accordance with determinant reference of Eo gene sequence, the CCSYD strain also belongs to the subtype I b. Eo gene also can acted as subtype basis of BVDV gene.4 pMD18-T/Eo cloning plasmid was cut at BmH 1 and Xho 1 for Eogene. Then, Eogene was sub-cloned to pET28a at the same enzymes cut point to establish prokaryotic expressed plasmid pET28a/E0. The plasmid was transplanted into Eoli BL21 （DE3）, which was identified with enzymes cut and PCR and the positive germ was conducted for IPTG induced expression. The result showed that prokaryotic expression plasmid pET28a/Eo was established successfully. While induced by IPTG, the recombinant germ could express aiming protein that accounts for 9.25% of the total germ protein.5 pMD18-T/E0 cloning plasmid was cut by BmH 1 and Xhol to obtain Eogene. Then, Eogene was sub-cloned into cloning vector pVAXl in the same enzyme cut point. Eukaryote expressed plasmid pVAXl/Eo was established and expressed by vesicle transinfection BHK-21. The result showed that the aiming gene was translated in BHK-21 cell, whichtested by RT-PCR. Indirect ELISA test indicated that eukaryote-expressed plasmid PAX1/EO could express aiming protein in vitro eukaryote cell.6 Gene vaccine of sika BVDV immunes rabbit in different doses and different times, the antibody response level was tested by indirect ELISA and cell immune response was tested by transformation test of lymphocytes and compared with the isolated strain and C24V of BVDV. The results showed that gene vaccine of sika BVDV produce not only cell immunity but also humoral immunity. The high dose group had the higher level of humoral immunity and cell immunity than the lower doses group. The times of immunity had no effect on both cell immunity and humoral immunity. Gene vaccine （immunity dosage >lmg/ml） produced the antibody level reached the peak serum titre in day 42 and immunity response of gene vaccine group is higher than inactive vaccine CCSYD and C24V, but it had the opposite result before 28th day. The cell immunity response of gene vaccine was lower than inactive vaccine （CCSYD, C24V）.7 The maximum security concentration of ribavirin, astragalus membranaceus, herbal bouttuyniac, interferon, curcumae, rhizoma coptidis were tested in MDBK cell that was cultured in vitro by utilizing tissue and cell cultivation. Moreover, the effect of the 6 medicines to MDBK cell infected by BVDV was tested, and the sensibility medication of BVDV was selected. The results showed that the maximum security concentration （lowest degree of dilution） of all kinds medicines to MDBK cell undamaged were: ribavirin（214）, astragalus membranaceus（23）, herbal houttuyniac（28）, interferon（25）, curcumae（2 ）, rhizoma coptidis（27）, respectively. The degree of three anti-BVDV medicine methods is: curcumae>herbal houttuyniac> astragalus membranaceus>interferon>ribavirin> rhizoma coptidis.更多还原