选取了黑熊的9个微卫星基因座(ABB1、ABB3、ABB4、ABB5、ABB6、ABB7、ABB10、ABB11、ABB12)进行遗传多样性研究.结果:东北黑熊养殖群体的9个微卫星座位共检测到60个等位基因,平均为6.67个;9个微卫星基因座的平均多态信息含量为0.589 7,其中6个微卫星基因座的多态信息含量(PIC) ＞0.5,9个微卫星基因座的平均观察杂合度、平均期望杂合度分别为0.536 3、0.625 2.由此证明,9个微卫星标记均具有高度多态性,可用于东北黑熊的遗传多样性分析.

布鲁菌病(brucellosis)是由布鲁菌引起的人兽共患传染病,家畜牛、羊、猪最常发生,且可传染给人和其他家畜,其特征是生殖器官和胎膜发炎,引起流产、不育和各种组织的局部病灶.本病广泛分布于世界各地,目前在我国人畜间仍有发生,给畜牧业和人类的健康带来严重危害[1].

According to the published gene sequence of goose parvovirus(GPV) strain B in GenBank,a pair of specific primers with Xho I and BamH I restriction enzyme sites were designed and synthesized.The VP1 gene fragment of GPV was amplified by PCR and cloned into pMD18-T simple vector.The fragment was subcloned to eukaryotic expression vector pVAX1 and the recombinant plasmid pVAX1-VP1 was identified by PCR,double restriction enzyme analysis and sequence analysis.Then the recombinant pVAX1-VP1 was transfected to Vero cell with Lipofectamine 2000.The transfected cells were detected by the indirect fluorescent antibody.The specific fluorescence could be observed on the cell surface.The 1 163 bp DNA fragment was amplified from RNA extracted from the Vero cell transfected with pVAX1-VP1 by RT-PCR.

To investigate the biological characteristics of YBLJ VP3 gene of goose parvovirus(GPV),according to the cloned gene sequence of GPV YBLJ strain in GenBank(JN836326),a pair of specific primers with Hind Ⅲ and XhoⅠ sites were designed.VP3 gene was amplified by PCR,and subcloned into pcDNA 3.1.The recombinant plasmid was transferred into Vero cells with Lipofectamine 2000,and its expression was detected by RT-PCR and indirect immunofluorescent assay(IFA).The results showed that the eukaryotic expression vector of pcDNA-VP3 was successfully constructed,a 1 605 bp specific band appeared in the experimental group by RT-PCR,the expression of VP3 was detected by IFA,which laid a foundation for development of GPV nucleic acid vaccine.

According to GenBank published gosling plague virus strain B gene sequence,specific primers were designed for cloning a 1 457 bp gene fragment.The fragment was cloned to the vector pMD-18T simple,then subcloned to prokaryotic expression vector pGEX-4T-1,and constructed a prokaryotic expression plasmid pGEX-VP3.The target protein was expressed in E.coli and induced by 0.1 mmol/L IPTG.The Western-blot analysis showed that the protein has a good reactivity.This is relevant for the development of genetically engineered vaccine and has laid a solid foundation.

本试验根据GenBank上发表的鹅细小病毒B株基因序列,针对vp3基因设计并合成了一对特异性引物,建立GPV vp3基因的PCR诊断方法。结果表明,该方法扩增的产物与预期大小相符(约654bp),而其他相关病毒均为阴性反应,敏感度为12.4pg。对15份GPV疑似病例检出的阳性率为100%,而琼脂扩散试验(AGP)检出的阳性率为60%。证明建立的PCR方法特异性强、敏感性高,可用于临床病料的快速诊断。

In order to establish the method for detecting GPV,research rabbit anti-GPV IgG was used to be capture antibody and anti-GPV monoclonal antibody was used to be detection of antibody.Based on chess-board experiment, the optimal coating concentration of rabbit anti-GPV IgG is 16 mg/L,the optimal working concentration of anti-GPV monoclonal antibody is 10.8 mg/L,The optimization of dilution for enzyme-labeled second antibody is 1:4 000,and double sandwich ELISA whose sensitivity of detecting GPV is 0.312 mg/L.was established.Dead gooses in a goose ranch in Yanbian were detected by this method.The positive rate is 75.79%.The concordant rate with virus neutralization test is 91.11%.Double sandwich ELISA established by this research provides a convenient and fast sero-diagnosis for detecting GPV and epidemiologic survey in district.

Balb/c mice were immunized with purified goose parvovirus(GPV) VP3 to prepare the monoclonal antibody.The anti-GPV hybridoma cells(4E5)were prepared with hybridoma technique,indirect ELISA and limiting dilution assay for 3 times were used to obtain the monoclonal antibody hybridoma cells.After identification,the MAb is IgG1 subtype and the light chain is κ.The results of Western blot showed that GPV VP3 protein could be specially recognized by 4E5 MAb.The anti-GPV VP3 monoclonal antibody was prepared successfully,which lay a foundation for diagnosis and treatment of GPV.

In order to prepare the monoclonal antibody of goose parvovirus,BALB/c mice were immunized by concentrated GPV and anti-GPV hybridoma cells(4B4) were obtained by hybridoma technique,indirect ELISA and limiting dilution assay.After identification,the heavy chain of McAb was IgG1 subtype and the light chain was κ chain.The results of ELISA and Western blotting showed that GPV and GPV-VP3 protein could be specially recognized by 4B4 McAb.It indicated that the epitope recognized by 4B4 McAb might be the conservated region of GPV VP3 protein.This research settled the foundation for the characterization of GPV epitope and the investigation of GPV diagnostic reagent.

为了研究鹅细小病毒灭活苗、亚单位疫苗、核酸疫苗3种疫苗对Balb/c小鼠的免疫效果,试验自制了这3种疫苗,对Balb/c小鼠进行3次免疫,应用间接ELISA方法测定体液免疫水平。结果表明:用小鹅瘟灭活苗、小鹅瘟VP3基因亚单位疫苗免疫Balb/c小鼠,在三免后第21天抗体的P/N值达到峰值,分别为8.21,3.56;小鹅瘟VP3基因核酸疫苗在三免后第28天,抗体的P/N值达到峰值,为2.09。说明这3种疫苗中小鹅瘟灭活苗对Balb/c小鼠的免疫效果最好。