鸡Dazl基因的克隆及其亚细胞定位研究

为克隆鸡Dazl(Deleted in azoospermia-like,Dazl)基因CDS序列,通过构建eGFP标记的DAZL真核表达载体,实现该基因产物的亚细胞定位以探究其生物信息学功能.提取1日龄鸡睾丸总RNA,通过巢式PCR方法扩增出Dazl基因的CDS,构建真核表达载体pEGFP-C1-DAZL.采用LipofectaminTMLTX介导重组表达载体pEGFP-C1-DAZL转染CEF细胞,48 h后于荧光倒置显微镜下观察其表达定位,同时利用RT-PCR和Western-blot进一步鉴定eGFP-Dazl和蛋白水平的表达情况.结果表明:克隆出Dazl基因的完整CDS,长度870 bp,共编码289个氨基酸,与公布的鸡Dazl基因(GenBank登陆号:NM_204218)CDS区同源性达99％,编码氨基酸完全一致.酶切鉴定和序列分析均表明真核表达载体pEGFP-C1-DAZL构建成功.转染48 h后,RT-PCR和Western-blot分别检测到949 bp特异条带和59.7 ku的融合蛋白.荧光显微镜观察显示,融合蛋白(eGFP-Dazl)主要分布于细胞核.     

传染性支气管炎病毒S1蛋白在Vero细胞的表达与分布

将传染性支气管炎病毒（IBV）ZJ971 S1基因亚克隆到绿色荧光蛋白（GFP）表达载体pEGFP—C2中，成功构建重组表达质粒pEGFP—ZJ971-S1。重组质粒在脂质体的介导下转染Vero细胞，借助荧光显微镜在转染后4h观察到S1—GFP融合蛋白的瞬时表达。免疫细胞化学染色（ICC）结果显示，抗ZJ971 S1D蛋白单克隆抗体和鸡抗IBVZJ971全病毒血清特异性识别了S1基因转染细胞，表明S1蛋白在Vero细胞中得到有效表达。荧光显微镜观察和ICC均表明，S1表达蛋白主要分布在转染细胞的胞浆内，而胞核中未见分布，提示IBVS1蛋白内可能存在与病毒装配相关的细胞定位信号。     

小鼠腹腔巨噬细胞的分离培养及Ipr1基因真核表达载体的构建和表达

为了获取Ipr1基因全长编码序列,构建与EGFP基因融合的表达载体,观察小鼠Ipr1基因在小鼠巨噬细胞中的表达情况及对细胞生长状态的影响,试验通过RT-PCR方法获得Ipr1基因,将获得的Iprl基因片段与pEGFP-C1载体片段连接后获得真核表达质粒pEGFP-Ipr1,经PCR、酶切鉴定正确后,脂质体瞬时转染pEGFP-Ipr1至第5代的小鼠腹腔巨噬细胞内,以空质粒pEGFP-C1转染组作为对照,采用Western-blot检测Ipr1基因的表达并用荧光显微镜观察融合蛋白的表达情况。结果表明:研究构建了真核表达载体pEGFP-Ipr1并进行了Ipr1基因转染,通过Western-blot检测在大约50 ku处有目的条带,转染24 h后有绿色荧光蛋白表达,并经G418筛选获得稳定转染Ipr1基因的小鼠腹腔巨噬细胞株。说明试验获得了易于转染的小鼠腹腔巨噬细胞株,成功转染了Ipr1基因并得以表达。     

猪圆环病毒2型Rep蛋白在真核细胞中的表达与定位

为了构建Rep蛋白真核表达质粒，研究PCV2复制过程中的亚细胞分布。本研究借助PCR方法扩增PCV2的Rep基因，并将其亚克隆到真核表达载体pEGFP-C3中，并转染PK15细胞和Vero细胞。结果显示，重组的pEGFP—PCV2-Rep融合蛋白能够在PK15细胞和Vero细胞中表达。结论认为pEGFP—PCV2-Rep质粒转染PK15细胞和Vero细胞后48h，PCV2的Rep蛋白主要定位在PK15细胞和Vero细胞的细胞核。     

奶牛LAP基因真核表达载体的构建及其在COS-7细胞中的瞬时表达

根据GenBank上发表的牛舌抗菌肽(Lingual antimicrobial peptide,LAP)基因cDNA序列设计1对特异引物,从患乳腺炎的奶牛乳腺组织中以RT-PCR方法扩增LAP基因片段,将其克隆到pMD19-T Simple载体中测序。重组质粒经EcoRⅠ+BamHⅠ双酶切回收目的基因片段,亚克隆入增强型绿色荧光蛋白表达载体pEGFP-C1中构建重组融合表达质粒pEGFP-LAP,将其脂质体转染COS-7细胞,经荧光显微镜观察到融合表达的绿色荧光蛋白,采用RT-PCR检测到LAP基因在COS-7细胞中转录。pEGFP-LAP重组表达质粒的成功构建为进一步研究奶牛LAP基因的表达特性及利用基因工程技术防治奶牛乳腺炎奠定了基础。     

徐淮山羊LPL基因克隆及其表达产物亚细胞定位的研究

旨在克隆徐淮山羊脂蛋白酯酶(LPL)基因的cDNA,并通过EGFP融合蛋白实现该基因亚细胞水平的表达定位.本研究通过RT-PCR方法克隆徐淮山羊LPL基因cDNA,并初步进行生物信息学分析,构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-LPL.聚乙烯亚胺(PEI)介导pEGFP-C1-LPL转染NIH-3T3细胞,48 h后在荧光倒置显微镜下观察,并用RT-PCR方法检测LPL mRNA在细胞内的表达.结果,成功克隆出山羊LPL基因1 530 bp长的cDNA,完整阅读框大小为1437 bp,共编码478个氨基酸,GenBank登录号为GU082383.信号肽区域预测结果表明LPL蛋白含有一小段信号肽序列,其可能性为100%.信号肽酶切割位置在第23和24个氨基酸之间,其可能性达到65.9%.成功构建融合表达载体pEGFP-C1-LPL,并且RT-PCR显示转染后LPL在mRNA水平上表达明显.EGFP-LPL融合蛋白定位在NIT-3T3细胞外和细胞膜部分.结果表明,首次成功克隆出徐淮山羊LPL基因cDNA;重组质粒pEGFP-C1-LPL构建成功,并在NIH-3T3细胞中明显表达.     

FMDV2A介导的乳腺上皮细胞双基因表达载体构建及表达

构建FMDV 2A介导的人白细胞介素2基因(hIL-2)和增强型绿色荧光蛋白(EGFP)基因双顺反子乳腺特异性表达载体,并验证其在乳腺上皮细胞中的表达.克隆奶山羊β-酪蛋白启动子序列,将hIL-2基因序列置于启动子之后,然后利用口蹄疫病毒2A(FMDV 2A)自剪切序列连接EGFP基因,构建出乳腺特异性的双顺反子表达载体pFIENβ,并利用脂质体转染山羊乳腺上皮细胞,然后用RT-PCR技术和Western blot检测hIL-2基因与EGFP基因的表达.重组质粒pFIENβ经酶切鉴定后表明构建成功,转染质粒PFIENβ和pEGFP-C1的细胞均观察到绿色荧光;从转染pFIENβ的乳腺上皮细胞中扩增出hIL-2基因和EGFP基因,而转染pEGFP-C1的细胞中只扩增出EGFP基因;经Western blot检测,转染pFIENβ的细胞中均表达了hIL-2和EGFP蛋白.结果表明山羊β-酪蛋白启动子能同时启动hIL-2基因和EGFP基因在山羊乳腺上皮细胞中的表达,并且利用FMDV 2A元件实现了hIL-2基因和EGFP基因的非融合型表达.     

猪瘟病毒EO基因在CHO细胞中的表达

将EGFPEO融合基因插入哺乳动物细胞表达载体pCI-dhfr中,构建重组质粒pCI-EGFPE0.将重组质粒转染CHO(dhfr'),在荧光显微镜下观察到少数细胞呈现绿色荧光,表明转染成功.通过MTX加压筛选后,在荧光显微镜下观察到多数细胞呈现强绿色荧光,表明重组融合蛋白EGFPE0在CHO细胞中得到大量表达.高效表达重组融合蛋白细胞克隆的获得为进一步大量制备可溶性的猪瘟病毒E0糖蛋白、制备其单抗筛选抗原表位及研究E0蛋白的生物学功能奠定基础.     

TGF-β1基因真核表达载体的构建及在猪脐静脉内皮细胞中的表达

应用RT-PCR技术从猪外周血单个核细胞扩增转化生长因子β1（Transforming growth factor-β1,TGF-β1）全基因,构建含有TGF-β1基因及EGFP报告基因的真核表达质粒pEGFP-C1-TGF-β1。采用脂质体法转染体外培养的猪脐静脉内皮细胞（SUVECs）后,通过直接荧光观察pEGFP-C1-TGF-β1融合蛋白在细胞中的分布定位,并通过RT-PCR、间接免疫荧光方法检测TGF-β1基因在SUVECs中的表达。结果在转染后1周观察到绿色荧光,RT-PCR、间接免疫荧光法检测TGF-β1表达均为阳性。本研究成功构建了含有绿色荧光蛋白基因的真核表达载体pEGFP-C1-TGF-β1,且TGF-β1基因在SUVECs中获得表达。     

鸡传染性法氏囊病病毒VP2蛋白GFP融合分子在COS7细胞内的表达

将鸡传染性法氏囊病病毒（IBDV）VP2蛋白基因插入增强型绿色荧光表达载体pEGFP-C1中,构建了真核表达载体pEGFP-VP2。PCR与酶切鉴定结果表明VP2基因成功插入到表达载体pEGFP-C1中。脂质体法转染COS7细胞后,荧光显微镜检测到了GFP-VP2融合蛋白的表达。用200 μg/ml的G418成功筛选到了稳定表达GFP-VP2融合蛋白的细胞系,表达蛋白经镍亲和柱得到了纯化。     

Cloning and expression of pigeon IFN-γ gene

This is the first paper describing the cloning of pigeon IFN-γ gene (PiIFN-γ) and the analysis of the in vitro expressed recombinant protein. The PiIFN-γ gene was identified by RT-PCR as a 498 bp, fragment coding for a precursor protein of 165 amino acids instead of 164 amino acids, as observed in the other avian species. The recombinant protein was expressed in vitro by an eukaryotic system and the biological properties of the cytokine were tested using a chicken macrophage cell line. The high degree of amino acid and nucleotide identity, shared with the ChIFN-γ, and the fact that the pigeon protein was functional on chicken cells, indicates a cross-reactivity between pigeon and chicken IFN-γ. The detection of the PiIFN-γ could represent an useful instrument in understanding the role played by this cytokine in immune response related to vaccinations and infectious diseases in the pigeon.     

喜树碱对NIH/3T3细胞TLR3表达及活化的影响

为研究喜树碱(CPT)对小鼠成纤维细胞NIH/3T3中Toll样受体3(TLR3)的表达及活化的影响,本实验利用CCK-8试剂盒测定了CPT在不同浓度下、不同作用时间对NIH/3T3细胞的毒性作用,并确定CPT对NIH/3T3细胞的基本无毒浓度为1μg/mL。通过流式细胞术检测CPT作用NIH/3T3细胞表明,TLR3平均荧光强度与对照组相比显著增强,表明TLR3表达增加。此外,ELISA试剂盒检测结果显示CPT作用后IFN-β表达量增加,并高于阳性对照组,差异极显著(p<0.01)。本研究表明CPT对NIH/3T3细胞的TLR3具有显著的激动作用。     

DINH与C3d3融合基因真核表达质粒的构建

为了构建DINH和mC3d基因融合的分泌型真核表达载体,试验将含有INHα(1～32)基因的pMD19-INH构建含有双拷贝INHα(1～32)基因的真核表达载体pcDNA-DPPISS-DINH,然后将鼠源C3d基因引入DINH基因3′端构建 了一种基于DINH-C3d3的抑制素基因疫苗pcDNA-DPPIS-DINH-C3d3.酶切及测序结果表明,pcDNA-DPPIS-DINH、pcDNA-DPPIS-DINH-sC3d3构建正确.     

Hepatic and intestinal CYP3A expression and activity in broilers

Cytochrome P450 is involved in drug metabolism. Subfamily CYP3A shows a degree of similarity across different animal species. However, little information is available about its expression and activity in broiler chickens. A RT‐PCR method was developed for the quantification of CYP3A37 expression in the liver and small intestine of broilers. A higher expression in the jejunum was observed compared with that in the ileum. In the liver, a significantly lower expression compared with that in the jejunum was noticed. Thus, the role of the small bowel in drug metabolism cannot be neglected in broilers. CYP3A activity was studied in vitro using midazolam as a substrate. Two protocols for the preparation of intestinal microsomes were compared. Mincing of the tissues before ultracentrifugation seemed to be more appropriate than a protocol based on ethylenediaminetetra‐acetic acid separation. CYP3A activity revealed to be the highest in the duodenum with a decreasing trend towards the ileum. Activity in liver was comparable to duodenal activity.     

Complete cDNA sequence and mRNA expression of dog preproendothelin-3

The full-length cDNA of dog preproendothelin-3 (PPET3) was cloned from lung tissue using RT-PCR and rapid amplification of cDNA ends. Aside from the poly (A) tail, the full-length cDNA was 1976 bp. A polyadenylation signal sequence and one copy of a consensus sequence, ATTTA, which is related to mRNA turnover, was found in the 3' noncoding region. The cDNA had a 594-bp open reading frame encoding a 198-amino acid polypeptide. Regions corresponding to a bioactive mature ET3 peptide, an intermediate form known as big-ET3, and an ET3-like peptide were observed in dog PPET3. Expression of PPET3 mRNA was detected throughout the organs examined, which included heart, lung, liver, kidney, spleen, stomach, pancreas, duodenum, colon, uterus, ovary and testis.     

JSRV-Env诱导转染NIH 3T3细胞后对VEGF和EGFR因子表达的影响

通过探索JSRV Env真核表达质粒诱导转染小鼠NIH 3T3(小鼠成纤维细胞)后对血管内皮生长因子(VEGF)和表皮生长因子受体(EGFR)表达的影响,进一步探讨其变化与绵羊肺腺瘤病(OPA)肿瘤发生的关联性。首先体外培养NIH 3T3细胞,设立转染pEGFP-C1-env、pEGFP-C1及未转染细胞组。本研究采用实时荧光定量PCR方法检测VEGF和EGFR mRNA水平上的表达;ELISA方法检测两细胞因子在不同处理的NIH 3T3细胞培养上清中蛋白的表达。结果显示,相比pEGFP-C1和未转染细胞组,转染pEGFP-C1-env细胞组中VEGF和EGFR mRNA的表达量均显著升高(P<0.05);且在该处理组的细胞培养上清中检测到VEGF和EGFR蛋白浓度呈明显上升趋势,差异均有统计学意义(P<0.05)。转染pEGFP-C1细胞组相比未转染细胞组VEGF和EGFR mRNA和蛋白表达量均差异不明显(P>0.05)。结果表明,JSRV Env在诱导转染小鼠NIH 3T3细胞转化过程中,通过qPCR和ELISA的检测结果均可得知VEGF和EGFR表达量均升高,由两者的过表达变化推测VEGF和EGFR在OPA肿瘤发生过程中可能起关键作用,同时为OPA针对两者进行的基因靶向治疗方法提供理论基础。     

刚地弓形虫GRA3基因的克隆与表达

采用PCR方法扩增弓形虫GRA3基因,将扩增产物与pMD18-T Simple载体连接,重组质粒经PCR、双酶切鉴定后测序;构建pGEX-4T-1／GRA3表达载体,经IPTG诱导表达后,进行SDS-PAGE、Western blot分析。结果显示,克隆的GRA3基因片段长687bp,含有1个669bp的开放阅读框,编码222个氨基酸,与GenBank中UK株（AF414079）的同源性为99．8％;表达的融合蛋白为50Ku,能被弓形虫阳性血清识别;表明该融合蛋白具有较好的免疫反应活性。     

鹅细小病毒VP3基因的克隆与表达

鹅细小病毒(Gooseparvovirus, GPV),主要引起雏鹅和雏番鸭感染发病,被感染禽的发病率和死亡率可高达70%～100%,是一种传播快、死亡率高的高度接触性传染病,亦称小鹅瘟.     

鹅细小病毒VP3基因真核表达载体的构建及其在Vero细胞中表达

根据已发表的鹅细小病毒(GPV)B株核苷酸序列,设计并合成了1对引物,PCR扩增GPV吉林分离株主要结构蛋白VP3基因,获得大小为1605bp的核苷酸片段。将该片段纯化后克隆入pMD18-T载体,转化感受态大肠杆菌JM109,双酶切鉴定,筛选阳性重组质粒并测序。经过酶切和连接反应,将VP3基因克隆入真核表达载体pVAX1,转化感受态大肠杆菌DH5α,筛选阳性克隆,提取质粒,进行了PCR和酶切鉴定。通过脂质体法将pVAX1-VP3转染Vero细胞,RT-PCR和间接免疫荧光法检测。结果显示,VP3基因克隆成功,与GPVB株核苷酸序列同源性为96.2%;PCR和酶切鉴定结果证实,成功构建了含VP3基因的GPV真核表达载体pVAX1-VP3。提取转染该质粒的Vero细胞RNA,RT-PCR扩增,在1000～2000bp可见一明显DNA条带;间接荧光抗体染色转染细胞,在细胞表面可见特异荧光。     

Xanthophylls increased HDLC level and nuclear factor PPARγ, RXRγ and RARα expression in hens and chicks

This study was designed to investigate effects of xanthophylls on serum lipid profile (triglyceride, TG; cholesterol, CHO; high‐density lipoprotein cholesterol, HDLC; and low‐density lipoprotein cholesterol, LDLC) and nuclear factor (peroxisome proliferator‐activated receptor gamma, PPARγ; PPAR gamma coactivator 1 alpha, PGC1α; retinoid X receptor gamma, RXRγ; and retinoic acid receptor alpha, RARα) gene expression of breeding hens and chicks. In experiment 1, 432 hens were divided into three groups and fed diets supplemented with 0 (as control group), 20 or 40 mg/kg xanthophylls. Blood was sampled at 7, 14, 21, 28 and 35 days of trial. Liver, duodenum, jejunum and ileum were sampled at 35 days of trial. Results showed that serum HDLC level of hens was increased after dietary 40 mg/kg xanthophyll addition for 21, 28 and 35 days, while serum TG, CHO and LDLC were not affected. Xanthophyll addition also increased PPARγ expression in jejunum, RXRγ expression in duodenum and jejunum, and RARα expression in liver and duodenum. Experiment 2 was a 2 × 2 factorial design. Male chicks hatched from 0 or 40 mg/kg xanthophyll diet of hens were fed diet containing either 0 or 40 mg/kg xanthophylls. Liver, duodenum, jejunum and ileum were sampled at 0, 7, 14 and 21 days after hatching. Blood samples were also collected at 21 days. Results showed that in ovo xanthophylls elevated PPARγ in duodenum and jejunum, and RXRγ and RARα in liver of chicks mainly within 1 week after hatching, while dietary xanthophylls increased serum HDLC level and PPARγ and RXRγ in liver from 2 weeks onwards. In conclusion, our research suggested xanthophylls can regulate serum lipid profile and nuclear factor expression in hens and chicks.