基因工程在动物医药学中的应用与研究进展

本文阐述了基因工程在动物医药学中的最新应用研究，重点介绍了基因工程药物、基因工程抗体、基因治疗技术、基因诊断技术等几大生物医药技术的研究和应用，在预防和临床医学上以及对传统医药学都产生了深刻的变革，具有广阔的应用前景。     

猪传染性胃肠炎病毒S基因核酸疫苗免疫小鼠体液免疫功能变化的研究

本实验对TGEV S基因核酸疫苗免疫后体液免疫功能变化进行了研究,同时比较了两种核酸疫苗单独免疫小鼠后体液免疫应答的变化,证明重组核酸疫苗诱导小鼠产生体液免疫应答。     

马鹿Zfx/Zfy基因的克隆及序列分析

根据GenBank中公布的牛Zfx/Zfy基因的mRNA序列,设计特异性引物,对马鹿Zfx/Zfy基因进行扩增,并对该序列进行分析。结果表明,克隆得到马鹿Zfx基因序列长为2 115 bp,GenBank登录号为KP257294.1,编码696个氨基酸;Zfy基因序列长为2 406 bp,GenBank登录号为KU041539,编码801个氨基酸。依据Zfx/Zfy基因氨基酸序列构建进化树,分析表明,马鹿Zfx基因与人的亲缘关系较近,与聚为一类的野牛、藏羚羊、虎、家猫、家牛、家犬、绵羊的亲缘关系稍远;Zfy基因与家牛、野牛、藏羚羊、绵羊的亲缘关系较近,与其他动物及人的亲缘关系较远。本研究首次克隆了马鹿Zfx/Zfy基因,为研究该基因蛋白质的结构和功能奠定了基础。     

Expression and functional analysis of the Follistatin-like 3 (FSTL3) gene in the sheep ovary during the oestrous cycle

Follistatin-like 3 (FSTL3) is a regulator of cellular apoptosis and was previously identified via RNA-Seq to be associated with follicular development in mammalian ovaries. However, the mechanism underlying the FSTL3 regulation of oestrus in sheep remained poorly understood. In this study, the oestrogen (E2) and progesterone (P4) concentrations in blood were detected, and the expression level and functional analysis of FSTL3 in the ovary were studied during the different reproductive stage in Aohan fine wool sheep (seasonal breeding breed in China). The concentrations of E2 and P4 at the anestrus were significantly lower compared to dioestrus, proestrus and oestrus stages. Higher expression levels of FSTL3 were observed in the sheep ovary, hypothalamus, and thyroid. During different reproductive stages, higher expression levels were found during the stages of dioestrus and proestrus, while lower levels were found during the oestrus and anestrus stages. Functional analysis of FSTL3 was performed in primary granulosa cells (GCs) of sheep. The concentration of E2 increased significantly after RNAi interference of FSTL3, while the P4 level decreased. FSTL3 can decrease P4 levels, which might be involved in mediating oestrous cycle in sheep.     

黄羽肉鸡ACSL1基因多态性与腹脂性状的相关性研究

长链酯酰辅酶A合成酶(ACSLs)是长链脂肪酸通过硫代酯化进而合成酰基辅酶A衍生物所必需的酶,也是脂肪酸代谢的第一步.哺乳动物ACSL家族由ACSL1、ACSL3、ACSL4、ACSL5和ACSL65个不同的成员组成,ACSL1是主要的异构体之一.为探讨黄羽肉鸡ACSL1基因作为腹脂性状分子标记的可行性,本实验采用PC...     

山羊ACSS2基因的克隆、序列分析及组织表达研究

本研究旨在对山羊乙酰辅酶A合成酶2（acetyl-CoA synthetase 2,ACSS2）基因进行克隆和生物信息学分析,并检测其在山羊不同泌乳时期乳腺组织中的表达量变化。以山羊乳腺组织RNA为模板,采用RT-PCR方法扩增并克隆山羊ACSS2基因完整CDS区序列,对测序结果进行生物信息学分析,并对ACSS2基因在山羊不同泌乳时期乳腺组织中的表达量进行分析。结果显示,山羊ACSS2基因CDS区序列长2 106 bp,编码701个氨基酸;山羊ACSS2基因与牛、马、人、犬、猪、小鼠和鸡的同源性分别为97.8%、92.0%、91.3%、91.3%、91.1%、88.1%和73.3%。蛋白理化性质分析结果表明,ACSS2蛋白分子质量为78.72 ku,理论等电点为6.03,属于酸性蛋白;跨膜结构和信号肽分析表明,ACSS2蛋白不含跨膜结构和信号肽;结构域分析表明,该蛋白含有1个乙酰辅酶A合成酶N端结构域。亚细胞定位分析结果表明,该蛋白主要分布在内质网（44.4%）、线粒体（33.3%）、细胞质（11.1%）和细胞核（11.1%）中。蛋白质结构预测发现ACSS2蛋白含有α-螺旋（29.10%）、延伸链（21.54%）、β-转角（9.84%）及无规则卷曲（39.52%）。实时荧光定量PCR分析结果表明,ACSS2基因在不同泌乳时期均有表达,其中在泌乳中期表达量最高,在干奶期表达量最低。本试验结果为进一步研究山羊ACSS2基因在脂质代谢过程中的功能及转录调控机制提供了参考。     

Differential expression of genes implicated in liver lipid metabolism in broiler chickens differing in weight

ABSTRACT

1. Lipid parameters and expression of ACACA, APOA1, CPT1A, FASN, FOXO1, LIPG, PPARα and SIRT1 genes involved in lipid metabolism were investigated in two groups of high (HW) and low (LW) weight broilers from the same strain.

2. Blood cholesterol and liver triglyceride levels were significantly increased in HW chickens compared to LW broilers, while other parameters, i.e. blood triglyceride, blood HDL/LDL, liver cholesterol and total liver fat showed no significant changes in either group.

3. The relative expression of ACACA, APOA1 and CPT1A genes was significantly lower in the liver tissues of HW broilers than in the LW group. The mRNA levels of these three genes showed a significant negative correlation with abdominal fat deposition and live weight of broilers. However, relative expression of FASN, FOXO1, LIPG, PPARα and SIRT1 hepatic genes did not differ among broilers.

4. It was concluded that, of eight hepatic genes implicated in lipid metabolism, only the expression of three (ACACA, APOA1 and CPT1A) were significant for fat and leanness within the same strain of chicken. Since reducing body fat is a major goal in the broiler industry, these data can provide fresh insight into the molecular processes underlying the regulation of fat deposition in broilers.     

BRCA1基因多态性及其与奶牛乳房炎抗性的相关性研究

为了研究BRCA1基因突变与荷斯坦奶牛体细胞数和体细胞评分的关系,试验通过对BRCA1基因外显子13、14进行克隆、序列比对和挖掘已有突变的方法确定该基因的多态位点,采用SNaPshot技术检测了BRCA1基因25025 T>A和46126 G>T突变位点在北京郊区荷斯坦奶牛群体中的分布,并对突变位点与体细胞数和体细胞评分进行了关联分析。结果表明,荷斯坦奶牛BRCA1基因2个位点均检测到3种基因型,其中25025 bp位点TT基因型为优势基因型,46126 bp位点GT基因型为优势基因型。25025 bp位点AA基因型个体体细胞数(P<0.05)和体细胞评分(P<0.01)都显著低于TT和TA基因型;46126 bp位点TT基因型个体体细胞数显著低于GG和GT基因型个体(P<0.05),但3种基因型个体体细胞评分无显著差异(P>0.05)。本研究结果初步表明,BRCA1基因25025和46126 bp位点可作为中国荷斯坦牛乳房炎抗性的标记辅助选择。     

Cloning of Buffalo AQP9 Gene and Analysing its Expression in the Buffalo Tissues

Cloning buffalo AQP9 gene and analyzing its expression in buffalo tissues.A pair of primers was designed according to the released bovine AQP9 sequences in GenBank,which was used to clone buffalo AQP9 gene.The AQP9 gene was amplified by RT-PCR,whose nucleotide sequence and protein structure were analyzed by bioinformatics methods.The expression of AQP9 in buffalo tissues was assayed by Real-time quantitative PCR.The expression of AQP9 gene in buffalo ovary and testis tissue was detected by immunohistochemical staining method.The results showed that the cloned ORF length of buffalo AQP9 gene was 888 bp,which coded 295 amino acids.The results of multiple sequence comparison showed that the nucleotide sequence of buffalo AQP9 shared 99%,90%,97% and 88% homologeous compared with that of Bos taurus,Sus scrofa,Ovis ariessis and Homo sapiens,respectively,while shared 99%,86%,97%,83% homologeous for amino acids,respectively.Phylogenetic tree analysis indicated that AQP9 gene was highly conservative in the evolutionary process.Real-time quantitative PCR results showed that AQP9 gene expressed in buffalo liver,lung,brain,skin,testis and ovary tissues with different levels,had the most abundant expression in liver,followed by in skin and testis,less observed in lung and ovary.The results of immunohistochemical staining showed that the expression of AQP9 protein varied with the development of buffalo ovarian tissue,and gradually enhanced with follicle development.In testicular tissue,AQP9 protein expressed in spermatocyte and leydig cells of developmental stage testis.These results indicated that we had successfully cloned buffalo AQP9 gene sequences.The expression and its function of AQP9 in buffalo ovaries and testes might play an important role in follicle development and spermatogenesis.