香蕉14-3-3蛋白基因Ma-14-3-3d的克隆及序列分析(英文)

[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.     

牛瑟氏泰勒虫P23基因的克隆与生物信息学分析(英文)

[Objective] The aim of this study is to provide basis for developing genetic engineering vaccine and diagnostic kit for Theileria sergenti infection. [Objective] P23 gene of Theileria sergenti was amplified from its genomic DNA by PCR amplification, and cloned into the pGEM-Easy vector; then the sequencing result was analyzed with bioinformatics methods. [Result] Whole length of the P23 gene from Theileria sergenti is 684 bp containing a 672 bp open reading frame. The deduced amino acid sequence (223 amino acid residues) contains a signal peptide of 19 amino acid residues and two fragments of transmembrane domains, with relative molecular weight of the 25.886 kD and with the pI of 9.22. The homology between the yielded sequence and Chitose of Theileria sergenti P23 gene(TS-Chitose type, D84446), Ikeda of Theileria sergenti P23 gene(TS-Ikeda type, D84447) reached 99% and 90%, respectively. The sequence has been accessed in GenBank(EU573168). [Conclusion] The protein encoded by the P23 gene has better stability and immunogenicity, thus can be used as the antigen candidate for preparing genetic engineering vaccine for Theileria sergenti.     

Cloning and Expression Analysis of Sucrose Non-fermenting-1 Related Protein Kinase （SnRK） in Cucumis sativus L. Under Low Nitrogen Conditions

The sucrose non-fermenting-1 related protein kinase（SnRK）, whose expression is induced by kinds of hyperosmotic stresses, plays a key role in improving stress resistance of plants. In order to investigate the molecular mechanism of low nitrogen resistance in cucumber, the full-length cDNA of SnRK gene was cloned in this study. The result showed that SnRK gene was 1 548 bp in length, encoded 515 amino acids, and had more than 80% homology with other crops. The protein encoded by this gene was an unstable and hydrophilic protein with no transmembrane structure and no signal peptide. Under nitrogen-free conditions and low nitrogen conditions, the expression pattern analysis of SnRK gene showed that this gene was up-regulated and its expression increased and was significantly higher than the normal level as the nitrogen concentration decreased. In addition, the expression of SnRK gene was also inhibited in the high nitrogen level and was significantly lower than the normal level. The result of this study would help us understand the molecular mechanism of low nitrogen resistance in cucumber.     

棉铃虫蛋白酶体β5基因Habeta5的克隆与序列分析(英文)

[Objective] Using molecular biotechnology to clone the proteasome β5 gene from cotton bollworm (Helicoverpa armigera), this research aimed to provide basis for further research on the function of proteasome β5 gene in cotton bollworm. [Method] Total RNA was extracted from midgut of cotton bollworm. The full length cDNA of Habeta5 gene was cloned by using rapid amplification of cDNA ends (RACE) technology, then sequence analysis was carried out. [Result] The full length cDNA sequence was successfully cloned and isolated, named as Habeta5. It was 947 bp in length, contained an ORF (843 bp) and encoded 280 amino acid residues, with the predicted mass of 30.87 kD and isoelectric point(pI) of 9.60. In the deduced amino acid sequence, a proteasome β5 subunit domain lies between 74th to 261st amino acid residues. It has more than 62% identity to other insects such as Drosophila melanogaster. The proteasome β5 subunit conservative regions were very similar with each other. Molecular evolution by Neighbor Joining method indicated that Habeta5 was homologous with other proteasome β5 subunit of species. [Conclusion] Sequence alignment shows that the cloned fragment is a proteasome β5 subunit gene (GenBank accession number: FJ358434).     

四川白鹅白细胞介素-2基因的克隆及生物信息学分析(英文)

[Objective] The study aimed to clone interleukin-2(IL-2) gene from Sichuan white goose. [Method] Based on the IL-2 gene of duck accessed in GenBank, a pair of primers was designed for cloning IL-2 gene from total RNA of peripheral blood lymphocytes of Sichuan white goose stimulated by ConA via RT-PCR technology. The yielded fragment was sequenced for bioinformatics analysis. [Result] The full length of IL-2 gene of Sichuan white goose is 468 bp that contains a 441 bp open reading frame(ORF), encoding 146 amino acid residues. Bioinformatics analysis shows that the amino acid sequence of IL-2 gene of Sichuan white goose contains four phosphorylation sites, a glycosylation site and a signal peptide with 21 amino acid residues. Homologies of IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and duck, chicken, turkey are 92.7%, 77.5%, 78.2% and 85.8%, 65.5%, 64.1%, respectively. By contrast IL-2 nucleotide sequence and amino acid sequence between Sichuan white goose and mammalian and rodents such as human, monkey, rat, bovine, horse, pig, cat, mouse, rabbit and deer, are all less than 45% and 28%, respectively. [Conclusion] The IL-2 gene of Sichuan white goose has closer genetic relationship with those of chicken and duck.     

马MxA基因第12外显子多态性检测(英文)

[Objective] The study aimed to establish a fast and accurate method to detect the polymorphism of the 12th exon of equine MxA gene. [Method] The 12th exon of MxA gene was amplified by mismatch PCR and the products were analyzed by restriction fragment length polymorphism (RFLP) to determine the point mutation at the 1 790 nt of MxA cDNA. The sequence of the PCR products was also analyzed. [Result] There were three genotypes (AA, AB and BB) in the 12th exon of equine MxA gene; the 2 081 nt of MxA cDNA mutated from G to C, correspondingly changing the 562th amino acid of the coding region of MxA protein from tryptophan to cysteine; the specific sequence of the PCR products amplified by mismatch PCR-RFLP was consistent with the analysis results of RFLP. [Conclusion] The mismatch PCR-RFLP was an easy method with accurate results to detect the polymorphism of the 12th exon of equine MxA gene.     

Porcine Interleukin-2 Expression in Insect Cells and Its Enhancement of Pig Immunity to Swine Influenza Virus Inactivated Vaccine

Mature porcine interleukin-2 （pIL-2） gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of the PH promoter. Recombinant plL-2 （rpIL-2） expressed in Sf9 insect cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunofluorescence assay. Western blot analysis confirmed that the rpIL-2 protein had a molecular mass of 20 kDa, which was larger than the molecular mass of the mature protein predicted based on its peptide sequence. The rpIL-2 protein induced in vitro proliferation of ConA-stimulated porcine splenocytes and enhanced in vivo protective immune responses induced by vaccinating the pigs with inactivated oil emulsion vaccine against swine influenza virus. The results showed that the rpIL-2 expressed in Sf9 insect cells has immunoenhancement effects; the finding lays the foundation for the preparation of a specific recombinant IL-2 protein and the development of a novel immune adjuvant of vaccines against various infectious porcine pathogens to increase the immunoprotective efficacy of vaccines.     

野桑蚕中肠组织cDNA文库的构建及丝氨酸蛋白酶基因片段的克隆与序列分析(英文)

[Objective] The aim of the study is to construct cDNA library of midgut tissue of wild silkworm and isolate the serine protease gene. [Method] The midgut tissue-specific cDNA library of wild silkworm was constructed via cDNA Library Construction Kit (TaKaRa), then the serine protease gene was cloned via sequencing of the yielded cDNA library. [Result] The titer of cDNA library reached 6.2×105 pfu/ml, average insert size was about 1.2 kb. The serine protease gene cDNA fragment was obtained from colony sequencing (Accession No: EU672968). The nucleotide sequence of the cloned 854 bp fragment encodes 284 amino acid residues. Homology analyses showed some homology between putative amino acid sequence of the cloned fragment and amino acid sequences of serine proteases from other ten insects. [Conclusion] The results may avail to reveal the resistance of silkworm and wild silkworm to exotic intrusion.     

Adjuvant Effects of Recombinant Plasmids of Porcine IL-4 and IFN-γ to Cysticercosis cellulosae Vaccines in Mice and Pigs

To investigate the adjuvant potential of porcine IL-4 and IFN-γ in mice and pigs, the genes of porcine IL-4 and IFN-γ were cloned and the recombinant mammalian expression plasmids were constructed for in vivo expression of the cytokines. Adjuvant effects of recombinant expression plasmids of IL-4 and IFN-γ （pcDNA-IL-4, pcDNA-IFN-γ） co-administrated with Cysticercus cellulosae crude antigen or TSOL18 recombinant protein antigen have been carried out in mice and pigs, respectively. We have demonstrated that recombinant plasmids of the cytokines as an adjuvant could induce stronger immune response in mice and pigs. With the C. cellulosae parasite antigen, porcine pcDNA-IL-4 induced higher specific antibody of immunized mice than pcDNA-IFN-γ. But pcDNA-IFN-γ is significantly stronger than that of no adjuvant or empty plasmids with the antigen control group. For the TSOL18 recombinant protein antigen vaccine, pcDNA-IL-4 still had a stronger ability to enhance specific antibody in swine than pcDNA-IFN-γ （P 〈 0.01）, but the immune protective rate was lower in challenged pigs （only 68.7%）. Although pcDNA-IFN-γ showed lower specific antibody, the protection rate was very high （91%） than other group （P 〈 0.01）. This study indicated that the recombinant expression plasmids of porcine IL-4 and IFN-γ display stronger adjuvant effects to C. cellulosae vaccine, further research should be carried out for understanding of the interaction mechanism.     

三元杂交猪IL-18全基因的序列测定及分析

白细胞介素-18（IL-18）又称γ干扰素诱导因子（IGIF）,研究表明IL-18是一种重要的新型免疫佐剂分子。为探讨猪IL-18的免疫佐剂作用,该研究根据已发表的猪IL-18基因序列,设计并合成1对特异性引物,从猪脾脏中直接扩增得到猪IL-18全基因,并进行克隆和序列测定、分析。     

三元杂交猪IL-18全基因的序列测定及分析

[目的]为进一步研发新型抗病毒制剂和疫苗佐剂、有效控制猪重大传染病奠定基础。[方法]根据GenBank中猪白细胞介素-18基因（IL-18）序列,设计1对特异性引物,应用RT-PCR方法从河南三元杂交猪脾脏淋巴细胞中扩增猪IL-18基因,并进行克隆、序列测定和分析。[结果]猪IL-18基因全长为579 bp,编码192个氨基酸的无活性前体蛋白,但前体蛋白并不含典型的疏水信号肽,在第35位氨基酸残基处有1个潜在的白细胞介素1β转换酶（ICE）剪切位点,剪切后变为具有生物活性的猪IL-18成熟蛋白。序列分析结果表明,该试验获得的猪IL-18基因与已知猪IL-18基因序列同源性很高,均为96%以上,其推导的氨基酸同源性在98%以上。[结论]该研究成功构建了猪IL-18基因的重组真核表达载体并得到了具有生物活性的瞬时表达产物。     

猪白介素家族重要基因的克隆、表达及其结构与功能预测分析

 【目的】克隆、表达和预测分析猪白介素家族重要成员的结构与功能。【方法】以猪白介素基因家族为对象，通过对猪PBMC刺激诱导，提取RNA，采用RT-PCR技术克隆IL-4、IL-6、IL-18等重要功能基因，并进行原核表达和结构与功能分析。【结果】成功地克隆和表达了IL-4、IL-6和IL-18基因，序列分析发现，克隆的基因与NCBI/GenBank上登载的参考序列的同源性分别为99.25%、99.21%和100％，与其它各物种的基因及其编码蛋白间具有较大的种属差异性；在大肠杆菌中表达的IL-4和IL-6重组蛋白具有较高的生物活性；结构预测表明，这些蛋白分子均含有较多的磷酸化修饰、糖基化修饰（除IL-6）、蛋白激酶以及信号传导结合位点，其中IL-4、IL-6蛋白分子的螺旋化程度较高，均在60%左右，水溶性较低，但这3种蛋白的立体空间结构均为致密的球状，说明该结构特征是其多种生物学功能发挥的基础。【结论】本研究成功地克隆、表达和分析了猪白介素家族的IL-4、IL-6和IL-18等重要分子的结构与功能，为其进一步的生物学功能研究及其应用奠定了基础。     

五指山小型猪生长激素释放激素受体基因的cDNA克隆及序列分析

1材料与方法 1．1材料随机采集五指山猪品种耳组织样共30份,将其迅速放入液氮带回实验室,-70℃保存备用。 1．2试剂RT—PCRKit、PCR产物纯化回收试剂盒、重组质粒抽提试剂盒,均购自天根生物工程有限公司;SalⅠ、BamHⅠ、pMD18-TVector试剂盒、Ecoli DH5α均购自TaKaRa公司。     

鲤鱼白细胞介素10(IL-10)基因cDNA克隆及序列分析(英文)

[目的]获取鲤鱼全长IL-10(interleukin 10)cDNA,并对其序列进行分析。[方法]利用DD-RTPCR(differential display RT-PCR)方法获得差异表达IL-10cDNA片段,以地高辛标记做为探针,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行核酸杂交筛选,克隆鲤鱼IL-10全长cDNA,并对该序列进行序列分析和同源性比较。[结果]鲤鱼全长IL-10cDNA共1117bp,包含55bp的5’端非编码区,一个540bp的编码179个氨基酸的开放阅读框及522bp的3’端非编码区。其中,3’非编码区包含3个mRNA不稳定基序"ATTTA";该蛋白序列具有IL-10家族的典型序列特征;序列同源性比较表明,所获得的序列与GenBank上登录的鲤鱼IL-10基因同源性为89.1%。[结论]该试验为进一步研究IL-10在体内的表达方式、功能特点、调控机理及其在炎症反应和免疫应答中的作用机制奠定了基础。     

猪白细胞介素-8荧光定量PCR检测方法的建立

用RT-PCR技术从猪肺泡巨噬细胞总RNA中扩增出猪白介素（IL）-8基因的278 bp cDNA片段,并克隆到pGEM-T Easy载体中,构建成含有猪IL-8 cDNA片段的重组质粒。通过质粒的Real-time PCR方法,建立猪IL-8 cD-NA的Real-time PCR标准曲线。该方法特异性强、灵敏度高、重复性强,可用于猪IL-8 mRNA含量的定量检测。     

猪IL-18 mRNA定量RT-PCR检测方法的建立

根据GenBank中猪白介素(IL)-18基因序列,设计1对引物,用RT-PCR技术从猪肺泡巨噬细胞总RNA中扩增出其411 bp cDNA片段,将其与pGEM-T Easy载体连接,经转化、筛选阳性克隆、酶切与测序鉴定后,构建出含猪IL-18 cDNA部分片段的重组质粒。后用反向PCR技术构建出与扩增411 bp片段共用同1对引物,但缺失174 bp的竞争重组质粒。通过上述2种质粒的竞争PCR方法,建立了猪IL-18 mRNA的标准竞争曲线,得到其直线回归方程^y=0.583 2x-2.903 4(R2=0.9898)。     

鲤鱼白细胞介素-1β全长cDNA的克隆·鉴定及其差异表达分析（摘要）（英文）

[目的]进行鲤鱼白细胞介素-1β（IL-1β）全长cDNA的克隆、鉴定及其差异表达分析。[方法]利用DD-RTPCR方法获得差异表达cDNA片段,对有丝分裂原刺激的鲤鱼外周血白细胞cDNA文库进行筛选,克隆了鲤鱼IL-1β的全长cDNA,并进行了序列分析和差异表达分析。[结果]获得的阳性克隆含有1个大小为831bp编码276个氨基酸的完整开放阅读框。聚类分析表明,鲤鱼IL-1β氨基酸序列与日本鲤鱼紧密聚为一支,氨基酸序列的同源性达95%,之后聚类顺序依次为鲫鱼、斑马鱼、猪、牛、马、人和小鼠。差异表达分析表明,经有丝分裂原刺激后前期（2h）白细胞中IL-1β的表达量显著增大,但随着时间推移（12,24h）并非一直较同期大,表达量总体趋势成峰形。[结论]为进一步研究IL-1β在体内的表达方式、功能特点和调控机理以及在炎症反应、应急反应和免疫应答的作用机制奠定了基础。     

猪Ob-Rb基因的克隆与分析

[目的]采用RT-PCR法克隆与分析瘦素受体06-Rb cDNA序列。[方法]从160日龄初情期的苏姜猪的下丘脑中提取组织总RNA．根据GenBank中猪06．RbmRNA全序列设计引物,用反转录-聚合酶链式反应（RT—PCR）进行cDNA扩增,获得1条343bp的片段,将PCR产物克隆于pGEM—Teasy载体后进行测序分析。[结果]用紫外分光光度计检测所提取的RNA质量,0D260／OD280=1.9,证明所提RNA的质量较好,无降解和蛋白质污染;用1％的琼脂糖检测所提RNA,有3条清晰的条带,分别是28S、18S、5S,28S与18S浓度比约为2：1．说明所提的RNA的完整性较好。所获D6-RbcDNA序列用Blast程序与在GenBank中发表的猪06-Rb cDNA序列（AF092422）比较表明,其同源性为100％,说明所扩增的序列是正确的。[结论]该研究为进一步探索Ob-Rb基因组织表达和作用机理奠定了理论基础。     

从Raji细胞克隆IL-10基因

[目的]探索一种简便可行的白细胞介素-10(IL-10)基因的克隆方法。[方法]以人Burkitt’s淋巴瘤细胞系Raji细胞为材料,提取培养的Raji细胞中的总RNA。根据GenBank中人IL-10基因序列设计1对特异性引物,用RT-PCR方法扩增人IL-10基因的编码cDNA。回收目的片段,将其与pMD18-T载体连接并转化到大肠杆菌感受态细胞DH5α,构建该基因的重组质粒。重组质粒经PCR和双酶切鉴定后进行测序。[结果]以骨髓细胞组织总RNA为模板进行RT-PCR,可从Raji细胞中获得了预期的约550bp特异性条带。成功构建了IL-10基因的重组质粒,PCR扩增和双酶切的鉴定结果说明该插入片段可能为IL-10cDNA。从获得的阳性克隆中挑选1个菌落来分析重组质粒的插入DNA片段序列。序列分析结果证实其与报道的IL-10基因的序列一致。[结论]该研究为IL-10的重组表达及其生物学活性分析奠定了基础。