Isolation And Identification On Sika BVDV And Cloning, Expression, Immunogenicity On Sika BVDV E₀ Gene

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.