Construction and Identification of Mammary Gland-specific Expression Vector of Bovine Tracheal Antimicrobial Peptide (TAP)

[ Objective] This study aimed to construct nmnmm_ry gland-specific expression vector of bovine tracheal antimicrobial peptide （TAP） gene. [ Method] TAP gene of dairy cattle was amplified from the mammary gland tissue by RT-PCR using a pair of primers which were designed according to bovine TAP cDNA se- quence （NM_174776） in GenBank, and then cloned into pMD19-T Simple vector for sequencing. The recombinant plasmid was digested using EcoRI and KpnI, the target gene fragment was recovered and inserted into general mammary gland-specific expression vector pBLG-EGFP harboring enhanced green fluorescent protein （ EGFP）, and transfected into bovine mammary epithelial cells （bMEC）, COS-7 cells and lactating rabbit mmmnary gland tissue by lipofectin transfection. The ex- pression of green fluorescent protein in transfected cells was detected under fluorescence microscopy, and the expression of TAP mRNA in rabbit mammary gland tis- sue was detected by semi-quantity RT-PCR. [ Result] The constructed mammary gland-specific expression vector pBLG-EGFP-TAP specifically expressed EGFP in transfected bMECs. In addition, semi-quantitative RT-PCR result showed that the expression level of TAP mRNA in rabbit mammary gland tissue was significantly enhanced after transfeeted with pBLG-EGFP-TAP. [ Conclusion] The mammary gland-specific expression vector pBLG-EGFP-TAP was successfully constructed, which provided important materials for further investigation of expression characteristics of TAP gene and prevention of bovine mastitis by using genetic engineering technology.

Preparation of Polyclonal Antisera of Dairy Cow S100A12 Protein

[Objective] This study aimed to prepare dairy cow anti-S100A12 antisem and develop a highly effective and sensitive immunological detection reagent for further investigation of the functions of dairy cow S100A12. [Method] Purified S100A12 protein was respectively emulsified with Freund＇s complete adjuvant and Freund＇s incomplete adjuvant as the antigen for immunizing New Zealand white rabbits to prepare the polyclonal antisera. The titer was detected using agar double diffusion assay and indirect enzyme-linked immunoserbent assay （ELISA） and the specificity was determined with Western Blot. [ Result ] The titer of anti- S100A12 antisera was 1： 8 as determined by agar double diffusion assay and over 1：409 600 by ELISA. Western Blot result showed that the polyclonal antisera could be specifically combined with S100A12 protein. [ Conclusion] The results indicated that anti-S100A12 polyclonal antibody with high fiter and high specificity was successfully obtained, which provided a novel tool for further investigation of the functions of S100A12 gene.

基于高通量测序技术分析四川两个奶牛场乳汁的菌群差异

【目的】本试验测定了两个奶牛场健康乳汁和乳房炎乳汁中微生物菌群的变化,以揭示不同奶牛场之乳汁菌群的异同,评估其对乳汁代谢的影响是否相同。【方法】采用16S rRNA高通量测序技术,分别测定两个奶牛场6头健康奶牛和6头乳房炎奶牛乳汁中微生物16S rRNA V4区序列,并对菌群群落结构和多样性进行比较,分析场内及场间的乳汁菌群差异。【结果】四组乳汁样本共获得4013234条原始序列,经过滤后获得2887024条优化序列。Alpha多样性Chao指数、Ace指数、Shannon指数、Simpson指数差异均不显著(P>0.05);Beta多样性四组样本均分别聚类;在场1和场2中,引起奶牛乳房炎的优势菌属分别是克雷伯氏菌属和埃希氏菌属;在2个奶牛场的健康乳汁中,场2的埃希氏菌属、葡萄球菌属的丰度显著高于场1;在2个奶牛场的乳房炎乳汁中,场2的埃希氏菌属、乳球菌属的丰度显著高于场1;2个奶牛场健康乳汁中的嗜冷菌总丰度分别为31.87%和38.72%;关联分析及功能预测分析表明,2个奶牛场健康乳汁与乳房炎乳汁优势物种之间的关系差异较大;场1无论是Level 1还是Level 2水平,均发现显著性差异的代谢通路,而场2均未发现显著性差异的代谢通路。【结论】本试验研究了两个奶牛场健康乳汁和乳房炎乳汁微生物菌群之间的异同,为两个奶牛场在乳房炎的预防工作以及原料奶在冷链运输过程中质量控制提供理论依据。

兔出血症病毒2型理化特性研究

【目的】本研究旨在研究该病毒的理化特性,评价不同处理条件对RHDV2的杀灭效果。【方法】本研究拟对临床疑似RHDV2感染致死的家兔进行RT-PCR鉴定病原,并利用不同pH值、不同温度、常用兽用消毒剂、不同浓度甲醛处理RHDV2,通过PMA-RT-qPCR对病毒理化特性进行研究。【结果】经RT-PCR检测与测序分析,确诊为RHDV2感染,该毒株VP60基因序列与GenBank中RHDV2(登录号:MN276176.1)一致性为98.35%。PMA-RT-qPCR结果表明,RHDV2对酸碱、高温、兽用消毒剂、甲醛均表现不同程度的敏感性。酸碱处理病毒,随pH值降低或升高,病毒杀灭率均有提高。65℃处理病毒30 min后,病毒杀灭率可达77.61%,83℃处理5 min后,病毒杀灭率可达95.10%。RHDV2对3种所测兽用消毒剂均敏感,其中聚维酮碘对RHDV2杀灭作用最好,作用30 min后,病毒杀灭率达88.78%。0.3%甲醛的杀灭率优于0.2%甲醛,作用30 min后,其杀灭率可达82.22%。【结论】本研究探究了RHDV2的理化特性,该病毒对酸碱、高温、兽用消毒剂、甲醛均有不同程度的敏感性。本研究为RHDV2的诊断、临床消毒剂的选择提供了参考依据,为该病毒致病机理研究与疫苗研发奠定基础。