猪肺炎支原体SC株P46基因的定点突变

猪肺炎支原体P46基因中有3个密码子TGA用于编码Trp而非终止密码子,为了在原核表达系统中进行猪肺炎支原体P46基因的根表达,根据其序列设计了3对引物,采用重叠延伸PCR(SOE-PCR)的方法将编码密码子TGA同义突变为TGG.将突变后的P46基因克隆至pEasy-T载体中,并进行测序.测序结果表明将P46基因中的TGA密码子突变为TGG很成功.     

Growth of differentiated ovine tracheal epithelial cells in vitro

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.     

肌肉生成抑制素基因的研究进展

肌肉生成抑制素(MSTN)属TGF-β超家族成员之一,主要分布在骨骼肌,对肌肉生长起负调控作用,MSTN的研究在畜牧业及医学领域具有重要意义.作者就MSTN的发现、分布、结构、生物学功能、作用机制及影响MSTN的因子如生长激素、生长分化因子11、T-cap蛋白、卵泡发育素、类胰岛素高亲和性结合蛋白、生肌决定因子等进行了综述,并展望了MSTN研究的发展趋势和应用前景.     

Analysis of reporter gene expression in ovine dermis and afferent lymph dendritic cells in vitro and in vivo

A flow cytometric technique was used to detect apoptosis and necrosis of bovine polymorphonuclear neutrophil leukocytes (PMN) using fluorescein isothiocyanate labeled annexin-V and propidium iodide (PI). Isolation of PMN from the blood following lysis by water or NH4Cl resulted in false positive results for apoptosis. Therefore, a method was developed to identify living, apoptotic and necrotic PMN simultaneously in a single 100 microl blood sample. To establish a positive control for PMN apoptosis, the effect of cycloheximide, actinomycin D, diamide, buthionine sulfoximine and sodium arsenite, that have been described to induce apoptosis by various mechanisms was tested. Only actinomycin D induced a significant increase in the percentage of apoptotic PMN after 2 h. Incubation of blood for 6 h with cycloheximide, actinomycin D and buthionine sulfoximine resulted in a significant increase of apoptotic PMN compared to control values. Sodium arsenite, mainly caused necrosis after 6 h of incubation.     

无痕基因敲除技术构建鼠伤寒沙门菌luxS基因突变株

为验证一种基于定点突变的无痕基因敲除技术(Scarless deletion)在鼠伤寒沙门菌中的有效性,本研究以鼠伤寒沙门菌中的lux S基因为靶基因,利用-Red重组酶和重叠延伸PCR(SOE-PCR)引入定点突变,以AAA代替lux S基因的起始密码ATG,改变lux S基因编码序列,通过测序验证并采用2型自诱导物(AI-2)试验检测是否有信号分子产生。基因测序结果显示lux S基因的起始密码ATG被引入的AAA代替;AI-2试验未检测到突变基因菌株的AI-2产物,而对照的野生菌株有AI-2产生,表明突变基因失活,方法有效。利用无痕基因敲除技术构建突变株简单快速,并且不影响被突变基因的另一条DNA单链序列,具有广泛的应用前景。     

Single-nucleotide polymorphism identification in the caprine myostatin gene

Polymerase chain reaction (PCR) products of MSTN gene amplified from 35 goats representing 17 Chinese indigenous goat breeds and five imported goat breeds were sequenced to identify the single‐nucleotide polymorphisms (SNPs) of a 379‐bp fragment including part of intron 2 and exon 3 of MSTN gene. A total of eight SNPs (A1980G, G1981C, A1982G, G1984T, A2121G, T2124C, G2174A and A2246G) were identified among the sequenced goats. The SNPs found are all located in intron 2 except for A2246G, which was a synonymous mutation in exon 3. Four haplotypes were sorted from these eight SNPs, of which, haplotype I (AGAGATGA) and haplotype II (GCGTGTAA) are the two main haplotypes with the frequency of 77.8% and 14.8% respectively. The SNPs found at positions 1980, 1981, 1982, 1984 and 2121 might be linked to inheritance completely.     

猪生成抑制素(MSTN) cDNA的克隆

从猪的肌生成抑制素(MSTN)编码序列中设计引物,以军牧一号猪肌细胞总RNA为模板,利用RT-PCR和嵌套PCR技术,扩增出MSTNcDNA片段.该片段全长1277bp,包含猪MSTN基因的全部编码序列.将所得片段与pMD18-T载体连接,转化到JM109大肠杆菌中,成功地筛选到阳性克隆,其质粒测序结果与文献报道的一致.经EcoRⅠ和PstⅠ酶解分析,cDNA片段与pMD18-T载体之间既有正向插入的克隆,也有反向插入的克隆,所得到的MSTNcDNA可用于原核和真核表达载体的构建.     

Effects of active immunization against myostatin on carcass quality and expression of the myostatin gene in pigs

The study was conducted to investigate the effects of active immunization against myostatin on the titer of myostatin antibody, carcass evaluation, activity of creatine kinase and the expression of the myostatin gene in pigs. Eighteen pigs were allotted into three groups (six pigs per group), and pigs in treatment 1, 2 and 3 were immunized with physiological saline, 1 mg or 4 mg myostatin per pig, respectively. Six pigs were killed by electrical stunning followed by exsanguination at BW of 100 kg. The results indicated that the titer of myostatin antibody was increased in treated groups compared to the control group on day 42 ( P  < 0.01) and d 84 ( P  < 0.01). The carcass lean percentage was significantly increased in the treatment groups compared to the control group ( P  < 0.01), and intramuscular fat was significantly decreased in the 4 mg group compared to the control group ( P  < 0.05). The muscle creatine kinase activity of pigs treated with 1 mg and 4 mg myostatin was lower than the control group. The immunization of myostatin signofocantly decreased the myostatin gene expression levels in muscle. It was concluded that optimal active immunization against myostatin could increase the content of myostatin antibody, suppress the activity of creatine kinase and the expression of myostatin gene, and therefore improve the carcass lean percentage for pigs.     

鸡肌抑素基因的原核表达及蛋白纯化

利用实验室保存的重组质粒pMD18-MSTN重新构建原核表达质粒pET-32a-MSTN,将其转化到宿主茵大肠杆菌BL21上并进行诱导表达,再将所表达的融合蛋白进行纯化.结果表明:该基因在宿主菌中成功表达;目的蛋白被成功纯化,且纯度较高.     

贵州香羊MSTN基因的多态性

肌肉生长抑制素（Myostatin,MSTN）是一种在骨骼肌中广泛存在的糖蛋白,是骨骼肌生长的负调控因子,其基因的缺失、插入或突变会引起肌细胞增生与肌纤维肥大从而提高产肉力。采用PCR-RFLP方法对67只贵州香羊MSTN基因进行了多态性分析。结果显示,5′UTR和外显子1没有出现变异,内含子1和外显子2中存在BspLⅠ、XmnⅠ、BmrⅠ、Hpy188Ⅰ4个酶切多态位点。纯合野生型（AA、CC、EE、MM）为优势基因型,杂合型（AB、CD、EF、MN）和纯合突变型（BB、DD、FF、NN）为非优势基因型,A、C、E、M等位基因为优势基因。     

IFITM3对羊口疮病毒在羊胚胎鼻甲上皮细胞上增殖的影响

羊胚胎鼻甲上皮细胞(OFTu)是羊口疮病毒(ORFV)高效感染和增殖的细胞,为分析ORFV感染对OFTu细胞干扰素诱导跨膜蛋白3(IFITM3)表达的影响及探讨IFITM3的表达状态对ORFV在OFTu细胞上增殖的作用,本研究对ORFV感染OFTu细胞后IFITM3基因的动态表达进行了分析,并通过构建IFITM3不同表达状态的感染细胞模型,探讨了IFITM3的不同表达状态对ORFV在OFTu细胞上增殖的作用。结果表明,ORFV感染可以诱导细胞IFITM3的表达,感染后2 h开始逐渐升高,感染后6～36 h与对照组相比差异极显著(P0.01)。过表达IFITM3的OFTu细胞会在感染后的24和36 h显著抑制(P0.05)ORFV的增殖,干扰表达IFITM3的OFTu细胞在ORFV感染后的前36 h促进了病毒的增殖,但差异不显著。研究结果进一步丰富了IFITM3的抗病毒谱,也为深入探索该蛋白的功能及作用提供了基础数据。     

牛双肌基因PCR-SSCP分析

双肌(肌肉生长抑制素)基因是一种肌肉生长的负调控因子,试验应用PCR-SSCP和测序的方法对我国秦川牛、南阳牛双肌基因的第3外显子进行了多态性分析.结果表明:第3外显子938位G→A的单核苷酸突变造成了南阳牛第3外显子扩增片段呈多态性;秦川牛中未发现突变.     

绵羊卵母细胞和卵丘细胞体外成熟过程中ghrelin mRNA的表达

运用实时定量RT-PCR技术检测了不同成熟培养时间绵羊卵母细胞和卵丘细胞ghrelin mRNA的相对表达量。结果表明,卵母细胞ghrelin mRNA相对表达量在16、24h呈现显著升高,而卵丘细胞ghrelin mRNA则随着成熟时间的延长表现显著降低。绵羊卵母细胞和卵丘细胞ghrelin mRNA的持续表达以及动力学变化提示这一新型分子在生殖功能上有潜在调控作用。     

Isolation,characterization, and in vitro differentiation of ovine amniotic stem cells

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.     

牛肌肉生长抑制素基因单核苷酸多态性分析

应用PCR-SSCP分析方法,对94头肉牛(公牛45头:西门塔尔34头,夏洛来11头;母牛49头:西门塔尔24头,夏洛来25头)的肌肉生长抑制素(MSTN)3个外显子进行了多态性分析.结果显示,第1外显子存在2种基因型,分别为AA型和AB型.经测序发现,第1外显子4 bp处存在C→G的碱基突变,导致编码的氨基酸由谷氨酰胺(Gln)→谷氨酸(Glu).统计结果表明,等位基因B的含量低,而且只在西门塔尔品种内含0.026 6.利用SPSS软件作最小二乘分析,结果表明,B等位基因与西门塔尔牛的成年体质量和犊牛出生体质量呈显著正相关.     

Factors influencing the degree of in vitro bacterial adhesion to ovine mammary gland epithelial cells

Bacterial adhesion to mammary gland epithelial cells (EC) may play a role in the pathogenesis of mastitis. In vitro adherence systems have been developed to study mastitis in cattle but little has been done in sheep. In this work, a method is described for obtaining mammary gland cell preparations containing greater than or equal to 65% EC from live or dead ewes, using a Ficoll-Hypaque flotation method (cell viability = 70-90%). An in vitro adhesion assay procedure was also developed to study the interaction between EC and ovine mastitis bacterial strains. It was observed that, under the test conditions, adherence increased as the incubation time was prolonged from 30 to 120 min (P less than 0.05). Adhesion was greater at incubation temperature of 37 degrees C than at 22 degrees C (P less than 0.001). An acidic pH (5.9) was associated with an increase in adhesion, when compared with a higher pH (7.2; P less than 0.05). Tween 20, Tween 80 and bovine serum albumin helped to eliminate a background of unbound bacteria from the test slides, but they also inhibited adhesion to some strains. Strain differences in adhesion and in ability to form a background were also observed. Some of these findings may have in vivo implications.     

牛肌肉生成抑制素基因真核表达载体的构建及其在COS-7细胞中的表达

将构建的牛pMD 18-T-MSTN克隆载体与真核表达载体pef-dhfr1a酶切,回收牛MSTN目的片段及pef-dhfr1a载体,构建了牛MSTN基因的真核表达质粒pef-dhfr1a-MSTN,然后转染COS-7细胞,将牛MSTN成熟蛋白编码序列在COS-7细胞中进行了表达.提取转染细胞的总RNA,采用RT-PCR和Western-blotting方法,分别从mRNA水平和蛋白质水平上检测到了牛MSTN基因在COS-7细胞中的表达产物,证明已经成功构建出该基因的真核表达载体.