乙醇脱氢酶应用研究现状

乙醇脱氢酶(ADH)是广泛分布于人和动物肝脏、植物及微生物细胞之中的含锌金属酶,具有广泛的底物特异性.ADH作为生物体内主要短链醇代谢的关键酶,近年来已经受到了国内外研究者的普遍关注,并对其同功酶的结构、理化性质、生物学功能、遗传学特性、分离提取技术等进行了较多的基础性研究工作,为其在科学研究和工农业生产中的应用奠定了良好基础.文章主要对ADH在工业分析、化工生产、食品和医药研究以及生物科学中的应用进行了概述,以便为其进一步研究开发提供参考.     

The Application and Research Progress of Chicken Embryo and Avian Cell Lines in Biomedicine

Chicken embryo has long been an important experimental model in basic and applied science because of its clear development process,especially in the early development of chicken embryo chorioallantoic membrane stage,due to its abundant blood vessels,it is a natural immunodeficiency host and can be used as an ideal experimental model for pathology,pharmacology and oncology research.The authors briefly described the tissue structure of early stage of chicken embryo,the application of chick embryo chorioallantoic membrane model in tumor,angiogenesis,organ transplantation,burn and other diseases,and the application of anti-cancer drug screening based on the pathological model of chicken embryo.The advances in the application of chicken embryo and avian cell lines in virus reproduction,vaccine production,therapeutic protein production and monoclonal antibody production were reviewed.A variety of human viruses,avian viruses and mycoplasma can proliferate in chicken embryos and avian cell lines and be used in vaccine production.In this paper,the development and characteristics of commonly used avian fibroblasts and pluripotent stem cells were described,and the source of commercial avian cell lines and some susceptible viruses were summarized.Chicken embryo expression system can produce human glycosylates at specific sites of target proteins,reduce the allergic reaction of the target protein to human,and poultry eggs are cheap and easily available,so it can be used as a suitable donor for the production of human monoclonal antibodies and therapeutic proteins.In this paper,the recent progress in the application of chicken embryos and avian cell lines in the field of biomedicine was introduced,and the future application of chicken embryos as animal models was forecasted.     

Genome-wide Association Study on Alive Litter Size Trait in Bama Xiang Pigs

In order to identify the molecular markers related to alive litter size of Bama Xiang pigs,the genome-wide association study (GWAS) was used to map and screen the candidate genes affecting the alive litter size trait.Ear tissue samples of 297 Bama Xiang pigs with multiple parity records were collected,and DNA was extracted and genotyped by porcine 50K SNP beadchip.After quality control and genotype imputation,the alive litter size of Bama Xiang pigs were GWAS by Tassel.The results showed that the average number born alive per litter of Bama Xiang pigs increased gradually with the increasing of parity in the range of 1-9 parities.A total of 32 816 SNPs were obtained after quality control and filtration.8 SNPs related to alive litter size of Bama Xiang pigs were screened by genome-wide association analysis,which were significant at genome or chromosome level.Based on the enrichment analysis of the coding genes in the region between 500 kb upstream and downstream of the associated significant SNP loci,and the QTL regions and gene functions related to porcine reproductive traits,4 genes (CAPZB,MSH3,CITED2 and HSD17B7) were finally identified to be candidate genes related to alive litter size of Bama Xiang pigs.     

Research Progress of Canine Coronavirus and Feline Coronavirus

Canine coronavirus (CCoV) and feline coronavirus (FCoV) belong to α-genus coronavirus of coronavirus family,porcine transmissible gastroenteritis virus (TGEV),porcine epidemic diarrhea virus (PEDV) also belong to the same genus.Genetic evolution analysis showed that different genotype of the virus could produce new variant strains through gene recombination,which caused great obstacles to the diagnosis and control of the disease.β-genus coronaviruses include bovine coronavirus (BCoV),canine respiratory coronavirus (CRCoV) and severe acute respiratory syndrome coronavirus (SARS-CoV).Among them,CRCoV has the highest homology with BCoV,but there are great differences in genomic structure,pathogenic mechanism and infection symptoms between this kind of coronavirus and α-coronavirus.CCoV and FCoV are widely spreading around the world,characterized by high morbidity and low mortality.Due to the characteristics of RNA virus and the influence of environmental selection pressure,the viruses continue to mutate and evolve,and new virulent strains appear one after another.The virulence of feline infectious peritonitis virus (FIPV) is greatly enhanced,some specific point mutations in the virus genome change the cellular tropism against the host.The pathogenesis of the virus mainly depends on the antibody-dependent enhancement (ADE) induced by virus infection.The epidemiological investigation and prevention and control of CCoV and FCoV should not only rely on the single factor of vaccine immunity,but also comprehensively consider the virulence of the virus,environmental conditions,pet self-immune resistance, and so on.The identification of CCoV and FCoV should be based on clinical symptoms,combined with routine hematological examination,serum biochemical examination and laboratory diagnosis techniques to prevent false positive and false negative results.     

禽网状内皮组织增殖病病毒的实时荧光定量PCR检测

基于Light Cycler平台,利用SYBR GreenI染料能特异与双链DNA结合而发荧光的特性,建立了一种荧光PCR方法检测禽网状内皮组织增殖病病毒(REV)的方法。整个检测过程2 h内可以完成,病毒的最低检出浓度为10-7,在特异性试验中,REV有特异性的扩增曲线,而禽流感病毒和阴性对照一样,没有特异性扩增。对REV患鸡各器官组织进行了荧光PCR检测,各脏器病毒含量从高到低依次是肝、脾、腺胃、肾、肌胃,肝脏中的病毒含量最高,肌胃中的病毒含量最少,肺中没有检测到病毒。基于SYBR GreenI的荧光PCR方法检测REV快速、敏感、特异性好,为开展REV病原学检测提供了一种快速、无污染的方法。     

抗盐酸克伦特罗单链抗体基因的克隆、序列分析及表达

提取分泌抗盐酸克伦特罗单克隆抗体杂交瘤细胞株的总RNA,通过RT-PCR技术,扩增VH和VL,用一段柔性肽链-(G4S)3将VH和VL连接成ScFv。测序后经NCBI Blast分析,所得ScFv具有重组功能性鼠抗体可变区基因的特征。将所得目的基因与pET-22b( )连接,转化E.coli BL21,用IPTG诱导表达,表达蛋白经SDS-PAGE分析,37℃培养5 h表达量较大,25℃诱导表达以可溶性为主。     

家蚕EST数据库的应用现状及展望

家蚕是重要的经济昆虫,也是鳞翅目昆虫的主要模式生物。表达序列标签(EST)在家蚕研究中的应用越来越受到关注。本文主要介绍了EST数据库的建立及其在家蚕研究中的应用现状,展望了EST在家蚕中的研究趋势。     

几种动物病原菌对替米考星耐药性的体外诱导

在测定替米考星对鸡毒支原体、多杀巴氏杆菌和猪胸膜肺炎放线杆菌的最小抑菌浓度基础上,采用药物浓度递增法体外诱导3种病原菌对替米考星的耐药性。结果鸡毒支原体经9次传代对替米考星产生了高水平耐药,多杀巴氏杆菌经7~9次传代对替米考星产生了高水平耐药,猪胸膜肺炎放线杆菌经14次传代对替米考星的耐受浓度未发生明显变化;鸡毒支原体和多杀巴氏杆菌替米考星诱导耐药株对红霉素、阿奇霉素、泰乐菌素、吉他霉素和林可霉素表现交叉耐药。研究结果提示,替米考星较易诱导鸡毒支原体和多杀巴氏杆菌产生耐药性,兽医临床应合理应用。     

马传染性贫血病毒驴白细胞弱毒疫苗株囊膜基因的优化及其DNA疫苗体外瞬时表达研究

马传染性贫血病毒(Equine infectious anemia virus,EIAV)的密码子使用频率与哺乳动物间存在着明显差异。为此,对马传染性贫血病毒驴白细胞弱毒疫苗株(DLA-EIAV)囊膜全长基因按照哺乳动物优势密码子的使用原则进行了重新设计和合成,并以此为基础通过重叠延伸PCR、限制酶切等方法得到结合型和分泌型囊膜基因,将其插入含有鸡beta-actin/兔beta-globin复合启动子(AG)的高效表达载体pCAGGS中,构建了EIAV驴白细胞弱毒疫苗株结合型和分泌型囊膜基因的DNA疫苗质粒pCAGGS-opti-bou-env、pCAGGS- opti-sec-env。将构建的质粒纯化后分别转染293T细胞,以间接免疫荧光和Western blot方法检测转染48 h后细胞及上清中囊膜蛋白的表达。结果显示,两种表达质粒均可正确表达EIAV囊膜蛋白,与相对应未优化的表达载体pCAGGS-wt-bou-env和pCAGGS-wt-sec—env相比,密码子优化的基因体外瞬时表达水平有极为显著的提高,而且蛋白表达部位也与预期的结果符合。这一结果为EIAV囊膜蛋白的单抗制备、表位鉴定、免疫试验、新疫苗的开发等奠定了基础。     

实时定量PCR技术在瘤胃微生物定量分析中的研究应用

随着对瘤胃微生物研究的逐步深入,一些以分子生物学技术为基础的定量分析方法对瘤胃微生物的研究方兴未艾。本文简要介绍了实时定量PCR技术的原理、操作程序以及该技术在反刍动物瘤胃微生物定量分析中的应用现状、取得的成果和前景展望。