IL-27p28基因多态性与中国朝鲜族人群哮喘的相关性

目的探讨IL-27p28基因启动子区g.-964 T>C、第2外显子区g.2905T>G和第4外显子区g.4730T>C 3个位点的多态性与中国朝鲜族人群哮喘的相关性。方法选取32例中国朝鲜族哮喘患者(病例组)和93名中国朝鲜族正常人(对照组)作为观察对象,采用PCR法扩增IL-27p28基因g.-964 T>C、g.2905T>G和g.4730T>C位点基因片段,单碱基延伸法进行基因分型,分析病例组和对照组的基因型频率及等位基因频率。结果病例组和对照组的基因型分布均符合Hardy-weinberg平衡定律(P>0.05)。g.-964位点的基因型频率及等位基因频率在病例组和对照组之间的分布差异有统计学意义(P<0.001);g.2905和g.4730位点的基因频率及等位基因频率在病例组和对照组之间的分布差异无统计学意义(P>0.05)。结论 IL-27p28基因多态位点g.-964 T>C可能与朝鲜族人群哮喘易感性相关,g.2905T>G和g.4730T>C多态性与朝鲜族人群哮喘未发现有相关性。     

乳腺生物反应器特异调控序列的构建

目的构建可以指导外源基因在动物乳腺组织特异表达的调控序列———牛α-S1酪蛋白基因调控序列。方法提取新生小牛肝组织的DNA,应用PCR分段扩增牛α-S1酪蛋白基因调控序列,经琼脂糖凝胶电泳鉴定后测序,再将各基因片段连接成完整的序列。并将外源基因ALB亚克隆入该调控序列内,转染C127细胞,经免疫组化法检测其表达情况。结果乳腺生物反应器特异调控序列———牛α-S1酪蛋白基因调控序列,包括其1.8kb上游序列、第一外显子、第一内含子、包含翻译起始位点的第二外显子和部分第二内含子,及其最后一个内含子的大部分、最后一个不翻译的外显子(含多聚A信号)及侧翼区全长5.8kb。位于该调控序列内的ALB高效表达。结论成功构建乳腺生物反应器特异调控序列———牛α-S1酪蛋白基因调控序列,此调控序列对外源基因的表达有正调控作用。     

西门塔尔牛瘤胃木聚糖酶基因的克隆及相关序列分析

目的克隆西门塔尔牛(Simmental)瘤胃木聚糖酶基因Rua11A,并进行相关序列分析。方法采用巢式PCR及单侧PCR技术从西门塔尔牛瘤胃微生物宏基因组DNA内直接克隆木聚糖酶基因Rua11A,并利用生物信息学软件对该基因序列及其编码氨基酸序列进行预测和分析。结果拼接后获得了1 278 bp的Rua11A编码区DNA序列,包括762 bp的木聚糖酶编码区、102 bp的连接序列编码区和411 bp的碳水化合物结合域(carbohydrate binding module,CBM)编码区,共编码425个氨基酸。氨基酸序列N-末端有1处富含疏水性氨基酸,不存在跨膜结构域;包含31个氨基酸的信号肽(signal peptide)及394个氨基酸的成熟肽(mature peptide),且理化性质稳定;预测的Rua11A空间结构具有11家族木聚糖酶的典型特征,属于CBM第四家族;含有11家族木聚糖酶高度保守的氨基酸片段及催化活性中心(catalytic active center)。结论成功克隆了西门塔尔牛瘤胃木聚糖酶基因Rua11A,并进行了相关序列分析,为Rua11A的异源高效表达及分子改造奠定了基础。     

鸡BF基因第2、3外显子序列及其多态性分析

目的分析鸡BF基因第2、3外显子序列及其多态性。方法从6羽霞烟鸡外周血淋巴白细胞(peripheral blood leukocytes,PBLs)中提取细胞DNA,以其为模板,经PCR法扩增BF基因的第2外显子、第2内含子和第3外显子,克隆至pGM-T载体上,构建重组质粒pGM-T-BF,并进行PCR、酶切和测序鉴定。利用生物信息学DNAStar软件将霞烟鸡与红原鸡第2、3外显子核苷酸及氨基酸序列进行多序列比对。结果经PCR、酶切及测序证明重组质粒pGM-T-BF构建正确。2种鸡BF基因序列的同源性可达88.6%~100%,第2、3外显子存在多态性变异位点91个,占总长度的17.04%,其中简约性信息位点66个,单变异位点25个;在编码相应的178个氨基酸残基中,多态变异位点49个,占总氨基酸残基数的27.53%,其中简约性信息位点37个,分单变异位点12个。结论鸡BF基因第2、3外显子呈高度多态性,且其多态性更多表现在氨基酸水平上。     

胃肠道间质瘤C-kit及PDGFRA基因突变检测及分析

目的 检测胃肠道间质瘤(gastrointestinal stromal tumors,GIST)患者中酪氨酸激酶受体基因C-kit及血小板源性生长因子受体A(platelet derived growth factor receptor A,PDGFRA)基因突变情况,探讨其与GIST发病的相关性。方法 从武汉大学人民医院及武汉大学中南医院收集的102例GIST患者石蜡肿瘤切片样本中提取DNA,采用PCR技术扩增特异性目的片段,并直接进行序列测定,分析样本中C-kit及PDGFRA基因突变情况。结果 在102份石蜡肿瘤切片样本中共检出C-kit基因突变76例,其中大多数突变发生于第11号外显子上,共65例,包括缺失突变43例,占66.15%(43/65),点突变12例,占18.46%(12/65),插入突变4例,占6.15%(4/65),点突变伴随缺失突变6例,占9.23%(6/65);7例样本存在第9号外显子突变;3例样本存在第13号外显子突变;2例样本检测出第17号外显子突变。共检出PDGFRA基因突变4例,其中第12号外显子突变的有3例,第18号外显子突变仅检出1例。结论 大多数GIST患者中存在C-kit基因的突变,且主要突变位置在第11号外显子;PDGFRA基因突变存在于C-kit未突变患者中。     

1份正反定型不符献血者标本的血清学及分子特性分析

目的对1份正反定型不符的献血者标本进行血清学及分子特性分析。方法采用吸收放散试验进行红细胞弱抗原检测,SSP-PCR方法进行基因分型,PCR产物克隆后进行测序分析。结果该标本血清学反应格局符合Ax亚型,分子生物学检测为A型。测序结果表明,Exon4含有2个连续碱基错义突变位点,分别为nt187G>A,nt188C>T,导致第63位氨基酸的改变(Arg63His)。结论该Ax亚型是由于Exon42个碱基突变,引起氨基酸的改变,导致糖基转移酶活性的降低,从而使红细胞上表达的A抗原大大减少。     

重叠延伸PCR法定点突变微生物产谷氨酰胺转氨酶基因

目的通过重叠延伸PCR法,定点突变微生物产谷氨酰胺转氨酶(Microbial transglutaminase,MTG)基因。方法根据GenBank中登录的Streptomyces sp.H197基因中MTG序列及重叠延伸PCR定点突变技术的原理,利用DNA-MAN5.0软件设计引物,以提取的Streptomyces sp.H197基因组DNA为模板,PCR扩增MTG基因,以扩增的MTG基因片段为模板,采用重叠延伸PCR技术对一个位点进行定点突变,胶回收PCR产物,与克隆载体pMD19-T连接后,转化感受态E.coli DH5α,提取质粒,经PCR及单、双酶切鉴定,并测序。结果重叠延伸PCR产物可见含有酶切位点和突变位点的特异性片段;重组质粒pMD19-T-MTG经PCR及单、双酶切鉴定,证明构建正确;测序结果表明,与野生型序列比对,仅有一个碱基发生改变,即第773位腺嘌呤突变为胞嘧啶,突变位点与预期一致,实现了目标位点的定点突变。结论重叠延伸PCR法对MTG基因序列预定位点突变成功,证明阳性克隆子含突变位点,为下阶段突变型功能表达奠定基础。     

成骨不全症患者LEPRE1基因突变位点的筛查

目的对COL1A1和COL1A2基因突变检测为阴性的成骨不全症患者进行隐性致病基因LEPRE1的筛查。方法采集成骨不全患者外周血样,提取基因组DNA,PCR扩增LEPRE1基因,直接测序法进行突变检测;采用PolyPhen、Align GVGD和SIFT突变功能预测软件分析突变对蛋白功能的影响。结果检测到1例成骨不全症患者在LEPRE1基因第5号外显子发生碱基GGA>AGA,发现甘氨酸被精氨酸替换的1个杂合突变位点(c.1045G>A,p.Gly349Arg);其母亲和外祖父有相同杂合突变,家庭其他成员和200份健康对照样本未检测到该突变;PolyPhen、Align GVGD和SIFT软件预测结果表明,p.Gly349Arg突变很可能影响蛋白的正常功能。结论 LEPRE1基因c.1045G>A突变很可能是成骨不全症潜在的致病突变位点。     

苯丙氨酸羟化酶基因突变家系PCR-SSCP分析

目的探讨聚合酶链反应-单链构象多态性(PCR-SSCP)检测方法对确定苯丙酮尿症(PKU)患者基因突变来源的应用价值,以便更好地应用于PKU基因诊断和产前诊断。方法对15例确诊的经典型PKU患者及其双亲的PAH基因的第3、5、6、7和11外显子及部分内含子行PCR扩增和非变性聚丙烯酰胺凝胶电泳分析;无PKU家族史的正常人作为正常对照。PCR-SSCP检测出异常外显子行PCR正、反向测序确定突变类型并分析患者的基因突变来源。结果5例患者的30个等位基因共检出16个突变,共14个突变等位基因来源均得到确定,1例患者(基因型为G247V/R243X)的突变来源未能确定。结论(1)PCR-SSCP检测方法可以确定PKU患者的基因突变来源(包括纯合突变和杂合突变),但对于2个基因突变位于同一外显子的杂合子患者,该方法不能很好地确定基因突变来源。(2)PCR-SSCP方法能够区分基因突变纯合子与基因突变杂合子。     

Ontogenic Expression Pattern and Genetic Polymorphisms of the Fatty Acid Transport Protein 4 (FATP4) Gene in Chinese Chicken Populations

In the current research, the polymorphism of FATP4 gene was analyzed in Erlang Mountainous chickens. A total of nine genetic variants were identified by FATP4 gene sequencing analysis across the chicken samples. Significant associations (p < 0.05) were observed for two SNPs (g.5608778C>T and g.5608814G>A in exon 6) with certain carcass traits (such as live weight, carcass weight, eviscerated weight) in S01 and S05 populations, respectively. Meanwhile, in S05 population, haplotype 3 (T-G) and haplotype 2 (C-A) were associated with higher and lower partial carcass traits such as live weight, carcass weight, eviscerated weight and semi-eviscerated weight, respectively. Moreover, we investigated the expression profile of this gene during ontogenesis in Mountainous black-boned chicken. Quantitative real-time PCR analysis showed that FATP4 mRNA had the highest expression level in small intestine tissue over all other tissues examined. The FATP4 mRNA levels presented remarkable developmental changes with age in the various tissues. These results suggested that the FATP4 gene might play an important role in controlling chicken carcass traits.     

产气荚膜梭菌β_2毒素基因的克隆及定点突变

目的　克隆产气荚膜梭菌 β2 毒素基因 ,并选择可能影响其生物活性的氨基酸的相关碱基位点进行定点突变 ,构建β2 毒素基因的突变体。方法　利用PCR从C型产气荚膜梭菌基因组DNA中 ,扩增出约 0 7kb的基因 ,将其克隆至pGEM T载体上。经GOLDKEY软件分析抗原表位 ,PCR定点突变技术进行 2 34位半胱氨酸→苷氨酸的定点突变。结果　β2 毒素基因核苷酸序列与报道的一致 ,其突变基因 6 99位核苷酸由T→G。结论　已成功克隆了 β2 毒素基因 ,并准确实施了预期定点突变。     

Effect of Porcine Somatotropin (pST) on Body Composition,Meat Quality and Characteristics of Backfat in Genetically Different Pigs

The influence of genotype, pST and stress reactivity on carcass composition, meat and fat quality was investigated in 57 growingfinishing pigs. Pietrain pigs contained more meat and less fat in carcass in comparison to Schwerfurter breed. Exogenous pST improved the carcass composition with regard to leaness. The inhibitory effect of pST on fat synthesis caused a reduced chemical fat in adipose tissue, reduced size and number of fat cells and an increase of relative content of polyunsaturated fatty acids. Halothane reactivity mainly influenced the composition of carcass. Stress-positive pigs were leaner than H'pigs. The factors pST, breed and halothane reactivity slightly affected the meat quality.     

Beneficial Effects of Natural Mineral Waters on Intestinal Inflammation and the Mucosa-Associated Microbiota

Natural mineral water (NMWs) intake has been traditionally used in the treatment of various gastrointestinal diseases. We investigated the effect of two French NMWs, one a calcium and magnesium sulphate, sodium chloride, carbonic, and ferruginous water (NMW1), the other a mainly bicarbonate water (NMW2) on the prevention of intestinal inflammation. Intestinal epithelial cells stimulated with heat inactivated Escherichia coli or H₂O₂ were treated with NMWs to evaluate the anti-inflammatory effects. Moderate colitis was induced by 1% dextran sulfate sodium (DSS) in Balbc/J mice drinking NMW1, NWW2, or control water. General signs and histological features of colitis, fecal lipocalin-2 and pro-inflammatory KC cytokine levels, global mucosa-associated microbiota, were analyzed. We demonstrated that both NMW1 and NMW2 exhibited anti-inflammatory effects using intestinal cells. In induced-colitis mice, NMW1 was effective in dampening intestinal inflammation, with significant reductions in disease activity scores, fecal lipocalin-2 levels, pro-inflammatory KC cytokine release, and intestinal epithelial lesion sizes. Moreover, NMW1 was sufficient to prevent alterations in the mucosa-associated microbiota. These observations, through mechanisms involving modulation of the mucosa-associated microbiota, emphasize the need of investigation of the potential clinical efficiency of such NMWs to contribute, in human beings, to a state of low inflammation in inflammatory bowel disease.     

Selective improvement of porcine fat firmness by a diet enriched in medium-chain fatty acids

The effects of 4% fat either rich in medium-chain fatty acids (MCFA; saturated fat) or in polyenoic fatty acids (PUFA; unsaturated fat) isoenergetically exchanging carbohydrates of a low-fat diet (control) on performance and product quality were evaluated with 180 growing pigs. Growth, carcass and meat quality were not affected by the fat treatments. Although elevated in blood serum, cholesterol was not increased in belly meat when the MCFA-rich diet was fed. Fatty acid composition of backfat reflected dietary fat composition. The significantly lowest shelf life and melting temperatures were found with the unsaturated-fat diet. When compared with control, fat tissue PUFA contents were slightly higher in the saturated-fat diet, and fat melting temperatures were somewhat lower. In contrast, the use of this MCFA-enriched diet increased penetrometer firmness in pure 4°C temperated backfat by more than 50% as compared with control and to about the tenfold level of the firmness obtained in the unsaturated-fat diet. Impression of flavour and odor in lean and fatty meat were not systematically affected by the diets. The present results show a high potential of MCFA-enriched diets to selectively increase fat firmness in pigs without greater undesired side effects on other traits of product quality.     

Variation der Fettbeschaffenheit im Rindfleischangebot

Variation of the Consistency of Fat in Beef Investigated was the fat of 313 young cattle, which is purchased by the Consumer on the fresh-meat-market. Among numerous calculated factors of influence it was determined that the sex of the animals, the amount of fatty tissue as well as the localization of the tissues on the carcass are the strongest causes of variance at the nutritional composition, refractometer value, hydroxyproline content, fatty acid pattern and size of fat cells of the samples. Growth-physiological traits and the mentioned criteria of the fat composition correlate loosely with the palatibility values of the prepared meat. The positive assessment of the meat at increasing fatty tissue proportion is to be opposite of the negative changes in the biological valuation of the fatty acids. Under the aspect of nutritional value and palatibility is a mean fatty tissue proportion on the carcass and the leaving of some fatty tissue on the muscles a recommendable compromise at the offer of meat.