Molecular cloning and expression of interleukin Ibeta (IL-1β) from red seabream (Pagrus major)

The interleukin 1β (IL-1β) cDNA was cloned from the red seabream (Pagrus major) by homology cloning strategy. A cDNA fragment was amplified by PCR using two degenerated primers, which were designed according to the conserved regions of other known IL-1β sequences, and elongated by 3' ends and 5' ends RACE PCR to get the full length coding sequence of red seabream IL-1β (RS IL-1β). The sequence contained 1252 nucleotides that included a 5' untranslated region (UTR) of 84bp, a 3' UTR of 410 bp and an open reading frame (ORF) of 759 nucleotides which could be translated into a putative peptide of 253 amino acids with molecular weight of 28.6 kD and putative isoelectric point pI of 5.29. The deduced peptide contained two potential N-glycosylation sites and an identifiable IL1 family signature, but lacked the signal peptide and the clear ICE cut site, which were common in other nonmammalian IL-1β genes. The RS IL-1β had the highest homology with piscine IL-1β according to phylogenetic tree analysis.     

水稻苯达松敏感致死基因的RAPD标记的克隆及测序

用3S Spin DNA Agarose Gel Purification Kit试剂盒将与水稻苯达松敏感致死基因相连锁的RAPD遗传标记S20—420和S316—600回收纯化，连接于pGEM—T载体并克隆测序，得到了S20—420和S316—600的全序列，其长度分别为423bp、606bp．将两端序列设计特异PCR扩增引物可用于检测水稻苯达松敏感致死基因和标记辅助育种．     

Molecular cloning and nucleotide sequence of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 paris of partially overlapping primers which span the entire genome of CSFV strain Shimen were designed and synthesized. Each cDNA fragment of strain Shimen was amplified by RT-PCR method from the anticoagulant blood of strain Shimen infected pig. The PCR fragments were cloned into pGEM-T vector respectively and sequenced. The results show that we have obtained the nucleotide sequence of strain Shimen. The viral RNA consists of 12 297 nucleotides including noncoding regions of 373 and 227 bases at the 5′ and 3′ end, respectively, and a single large open reading frame spanning 11 697 nucleotides in the middle, which encodes an amino acid sequence of 3 989 residues with a calculated molecular weight of 437.6×10³. The precisely sequencing of 5′ and 3′ termini is undertaking. Supported by the National Pandeng Project Huang Qianhua: born in 1968. Graduate student     

血小板反应素-1活性片段的克隆与表达

选择两段血小板反应素（TSP-1）中抑制血管再生的活性片段，设计两对引物，使用RT-PCR的方法从人血细胞中进行克隆并对获得片段进行测序验证．克隆片段大小分别为723和522bp，命名为聪P-1-1和聪P-1-2．利用原核表达载体pET-29a获得大肠杆菌重组子，重组子经过IPTG诱导以包涵体的形式表达相应的多肽片段．再对包涵体进行体外溶解、纯化，得到了目的多肽．     

极端嗜热厌氧纤维素分解菌基因文库的构建及其内切葡聚糖酶基因片段的克隆

以从云南邦拿掌温泉中分离、纯化的高温厌氧纤维素分解菌邦2菌(Cadicellulosiruptor)为材料,制备其总DNA,经限制性核酸内切酶EcoRⅠ部分酶切后,在T4DNA连接酶的作用下与经EcoRⅠ完全酶切、去磷酸化的质粒载体pUC18连接,然后转化E.coliJM109,建立了邦2的基因文库,经筛选鉴定得到6.3×103个重组子;重组子经刚果红平板验证:约有23.5%菌落呈现透明圈;重组子经EcoRⅠ酶切验证显示:重组质粒均含有外源DNA插入片段.结果表明已克隆到邦2菌纤维素酶系中的内切葡聚糖酶基因(ED基因)片段.     

同源重组在染色体DNA基因克隆中的应用方法

同源重组是生物界普遍存在的现象，从噬菌体、细菌到真核生物都有能发生同源重组的种类。如何把同源重组应用到基因克隆中成为近年来生物学家研究的热点。本文从宿主细胞的改造、重组功能的诱导、感受态细胞的制备、线性同源载体的获得及重组体的鉴定等几个方面具体叙述这种基因克隆的新方法，从分子水平上较为详尽地介绍了同源重组在染色体基因克隆中的应用、前景展望及存在的局限性。     

纳豆激酶基因克隆及其在大肠杆菌中活性表达研究

以纳豆芽孢杆菌基因组DNA为模板，PCR扩增了纳豆激酶基因（natto kinase gene）中编码前肽、成熟肽的核苷酸序列（pro-NK），构建大肠杆菌表达质粒pTYB102，转化大肠杆菌ER2566。在IPTG诱导下，分别在15℃（14h）、30℃（3h）、37℃（2h）条件下培养，pTYB102均能表达出有活性的纳豆激酶。实验证实纳豆激酶基因得到活性表达需要Pro序列。SDS－PAGE表明，15℃和30℃和37℃培养表达的杂蛋白更少。薄层扫描测定表达的纳豆激酶占菌体总蛋白30％以上。     

PMMA分子量及其结构的研究

本文分别采用粘度法，膜渗透法和凝胶渗透色谱法对国产溶液法ＰＭＭＡ分子量及其分布进行了测定，结合熔融指数数据并与国外产品进行比较，笔者认为，分子量、分子量分布和聚合物链结构是影响ＰＭＭＡ加工性能的重要因素。     

分子筛色谱柱快速活化方法的研究及应用

发展了一种机上快速活化分子筛色谱柱的方法。活化温度３００～３２０℃，全部活化时间只需要３ｈ，为常规活化方法的三分之一，活化效果优于现有方法。该方法用于探井井口气分析，取得了较好的经济效益。     

大熊猫IL-2基因的克隆与序列分析

本实验应用反转录-聚合酶链反应（RT-PCR）技术，从ConA诱导培养的大熊猫外周血淋巴细胞总RNA中扩增得到大熊猫IL-2基因，并将其克隆到PGEM-T载体中．经菌落PER鉴定、序列测定及序列分析，结果表明，经克隆得到的IL-2基因开放阅读框由465个核苷酸组成，编码一个由155个氮基酸组成的多肽，包括编码20个氨基酸的信号肽和135个氨基酸的成熟肽，该基因（已在Genbank中登录，序列号为：DQ852339）与已知的其他哺乳动物如犬、猫、人、猪、牛、羊、马、兔、鼠等的IL-2核苷酸同源性在66.5％（鼠）～91．5％（犬）之间；IL-2编码氨基酸同源性在54．8％（鼠）-85．2％（犬）之间；进化树构建结果表明大熊猫与犬亲缘关系最近．