人IL-16C端cDNA的克隆表达及初步鉴定

人 Ｉ Ｌ １６ 是近年报道的一个新的细胞因子,其 Ｃ 端 １３０ 个氨基酸的分泌型 Ｉ Ｌ １６ 广泛地参与了体内的免疫调节及炎症反应,并具有抑制 Ｈ Ｉ Ｖ 复制等多种功能．经从 Ｃｏｎ Ａ 刺激的人外周血单核细胞中分离总 Ｒ Ｎ Ａ,采用 Ｒ Ｔ Ｐ Ｃ Ｒ、分子克隆及序列测定的方法获得了 Ｉ Ｌ １６ Ｃ 端 １３０ 个氨基酸编码区的 ｃ Ｄ Ｎ Ａ,并在生物 素化融合蛋白表达系统中表达和纯化了该蛋白．结果表明：所得到的中国人 Ｉ Ｌ １６ Ｃ端的编码序列与国外报道的序列完全一致;已获得的 ３３ ｋ Ｄ 融合蛋白易于纯化,这一工作为进一步研究 Ｉ Ｌ １６ 的功能奠定了基础 ．     

新OPG／OCIF变体基因的克隆及其表达

从正常中国人肝细胞株Ｌ０２中抽提总ＲＮＡ,利用ＲＴ－ＰＣＲ技术克隆了亲破骨细胞抑制因子（ＯＰＧ／ＯＣＩＦ）ｃＤＮＡ,同时从人胎肾细胞株２９３细胞中克隆了ＯＰＧ／ＯＣＩＦ基因３′端基因组序列,序列分析结果表明,与国外文献报道相比较,中国人肝细胞株中该ｃＤＮＡ３′端存在一个赭石型终止码突变,从而使这一蛋白质所报道的在Ｃ端少８个氨基酸残基。从２９３细胞克隆的基因组序列也同样存在这一突变。将ＯＰＧ／ＯＣＩ     

人epiregulin在大肠杆菌中的表达

利用ＲＴ－ＰＣＲ的方法首次从人肺癌细胞Ａ５４９中扩增得到了ｅｐｉｒｅｇｕｌｉｎ的ｃＤＮＡ。在３’端添加６个组氨酸密码子后将期 克隆到本实验室构建的大肠杆菌高效碱性磷酸酶分泌表达系统中进行分泌表达。表达产物经Ｎｉ柱一步分离后得到纯化。对其进行氨基酸序列分析,结果与报道的人ｅｐｉｒｅｇｕｌｉｎ的序列一致。ＭＴＴ法测定其对Ｂａｌｂ／ｃ３Ｔ３细胞有很强的促增殖作用,而对表皮癌细胞Ａ４３１有较强的抑制生长作     

江浙蝮蛇磷脂酶A2基因的多样性研究

我们利用简并引物从江浙蝮蛇腺总ＲＮＡ经ＲＰ－ＰＣＲ扩增磷脂酶Ａ２（简称ＰＬＡ２）基因,并以碱性ＰＬＡ２（Ｂ－ＰＬＡ２）基因为探针,分离出了酸性ＰＬＡ２（Ａ－ＰＬＡ２）和两个未见报道的特征结构类同的基因,分别命名为Ａｓｎ＾４８－ＰＡＬ２和ＢＡ－ＰＡＬ２。双向测序测定了这组ＰＬＡ２同工酶（除信号肽外）基因的全序列,并由此推导编码的氨基酸序列。其中Ａ－ＰＬＡ２基因编码的氨基酸序列与较早报道的由蛇毒中分离     

人血管内皮抑制素在毕赤酵母中的表达纯化及活性测定

血管内皮抑制素是胶原ⅩⅧ C-末端的一个片段,是一个有效的血管生成抑制因子。本文克隆了人血管内皮抑制素的基因,并用毕赤酵母进行表达,表达量为50.5 mg/L。发酵液上清经SP Sepharose Fast Flow凝胶一步层析,产物纯度达到98%以上,纯化收率达到95%以上。毕赤酵母分泌表达的人血管内皮抑制素具有免疫活性;能明显抑制鸡胚尿囊膜新生血管的生长;并且能特异性地抑制bFGF刺激的人微血管内皮细的迁移,达到抑制效果为50%时所需的蛋白浓度（IC50）为0.4μg/ml。本研究为应用人血管内皮抑制素治疗肿瘤奠定了初步实验基础。     

抗草鱼出血病病毒多肽的结构分析

将草鱼出血病病毒（ＧＣＨＶ８７３）颗粒与随机噬菌体九肽库在体外作用,三轮筛选后,从３００个转化的单菌落中获得１６个与病毒高亲和力的噬菌体克隆。接着经过抗病毒试验获得６个能强烈抑制病毒复制的阳性噬菌体克隆,能使病毒ＴＣＩＤ５０下降５个数量级。通过对阳性噬菌体克隆随机插入区域的核苷酸序列分析,推导出多肽的氨基酸序列。发现６个能强烈抑制病毒复制的阳性克隆中多肽的氨基酸序列完全一致（ＮＨ２ＬｅｕＴｒｐＶａｌＧｌｙＧｌｙＧｌｙＡｒｇＡｓｎＡｌａ）,该结果提示多肽的抗病毒能力与多肽特异性氨基酸组成及结构有关。因此,抑制ＧＣＨＶ８７３复制特异性多肽的氨基酸序列的确定及结构分析,不仅为人工合成抗草鱼出血病病毒的多肽奠定了基础,同时也为抗病毒多肽制剂的研制提供了依据。     

豌豆卷须肌动蛋白Ⅱ类异型体cDNA克隆的序列分析

分析１５个豌豆卷须肌动蛋白ｃＤＮＡ 克隆的限制性内切酶图谱,发现在豌豆卷须中至少存在三类肌动蛋白异型体,分别命名为Ⅰ类（ＰＥＡｃ Ⅰ）、Ⅱ类（ＰＥＡｃ Ⅱ）和Ⅲ类（ＰＥＡｃ Ⅲ）异型体．三类异型体的克隆数目分别为１０、４和１个,表明三类异型体在豌豆卷须中的表达是不同的,很可能具有组织或发育阶段的特异性．对Ⅱ类异型体的三个ｃＤＮＡ 克隆ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１进行了全序列测定,所测定的序列已被ＧｅｎＢａｎｋ 数据库所接受．测序结果表明,ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１的序列长度分别为１５５０、１６８０和１０９１个核苷酸,其中编码区长１１３４个核苷酸,编码的氨基酸长度为３７７（ＰＥＡｃ１１缺少编码氨基端前９６个氨基酸的核苷酸序列）．三个克隆的核苷酸序列完全相同,差别仅在于３′非翻译区的长度不同,即ｐｏｌｙ（Ａ）的加入位点不同．这说明它们可能是由同一基因转录而来,但转录后的加工过程不同．豌豆卷须中肌动蛋白基因ｐｏｌｙ（Ａ）加入位点的使用,可能与组织或发育的特异性表达有关．此外,豌豆卷须肌动蛋白三类异型体之间的核苷酸序列同源性为８０％ ,氨基酸序列同源性为９４％ ．     

人脂多糖结合蛋白基因的克隆及序列测定

采用PCR技术,从人肝cDNA文库中扩增获得了1.5 kb的脂多糖结合蛋白（LBP）的全长基因.序列分析表明,克隆的LBP基因编码的氨基酸序列与文献报道相同.     

人血小板生成素 cDNA 合成、克隆及序列测定

利用ＲＴ-ＰＣＲ方法从中国人胎肝总ＲＮＡ中扩增出人血小板生成素（ｈＴＰＯ）的ｃＤＮＡ。序列分析结果表明,我们所获得的ｈＴＰＯｃＤＮＡ与文献报道中的基因序列高度同源,其中第４９７ｂｐ、５９５ｂｐ、７６７ｂｐ和７９５ｂｐ位碱基分别由Ｔ、Ｇ、Ｔ和Ｔ代替了文献中的Ｇ、Ａ、Ｇ和Ｃ,从而导致了１６６、１９９和２５６位氨基酸由报道中的Ｓｅｒ、Ｌｙｓ和Ｇｌｙ变为Ｐｈｅ、Ｇｌｕ和Ｖａｌ。     

人类黄体组织抑制素与激活素构型的研究

本文观察了抑制素／激活素α、βＢ与βＢ亚基基因在人早（３天）、中（３天）及晚（１５天）期黄体组织的表达。α－ｍＲＮＡｓ在早期黄体最高;βＡ－ｍＲＮＡｓ仅在早期出现,而βＢ－ｍＲＮＡｓ从黄体早期至晚期逐步增高。提示人黄体抑制素可能为抑制素Ｂ。晚期黄体可能有激活素Ｂ的生成。     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

中华眼镜蛇短链神经毒素cDNA的克隆及在大肠杆菌中的表达

利用RT－PCR技术从中华眼镜蛇毒腺组织中成功地克隆了短链神经毒素CDNA。测序结果表明,该基因开放阅读框架编码83个氨基酸残基,其中对个为信号肽,成熟肽为62个氨基酸残基。该基因与GenBank报道的相同物种的神经毒素基因有相当的同源性,不同物种之间的信号肽序列十分保守。将短链神经毒素CDNA再经PCR扩增除去信号肽序列,克隆到pT7ZZ表达质粒中,转化E.coliBL21（DE3）后,经IPTG诱导可高效表达分子量为23kDa②左右的融合蛋白。表达产物占菌体总蛋白的25％左右。     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

G蛋白Rab3a cDNA的克隆与表达

利用PCR法 ,从人胎盘总cDNA中扩增得到Rab3acDNA的全编码区 .序列分析表明 ,扩增得到的Rab3acDNA有 5个核苷酸发生了变异 ,但翻译的氨基酸与发表的完全一致 .将扩增得到的Rab3acDNA克隆于原核融合表达载体pGEX 4T 1中 ,在E .coliBL2 1中经IPTG诱导表达 .为了进一步鉴定表达产物 ,对纯化后的Rab3a蛋白进行了SDS PAGE、N端氨基酸测序、质谱分子量测定及氨基酸组成分析鉴定 .结果显示 ,表达蛋白的分子量约 2 5kD ,N端氨基酸序列为MASATDSR ,氨基酸组成分析表明 ,Rab3a蛋白获得了正确表达     

黑曲霉脂肪酶：基因克隆、表达及体外复性

根据黑曲霉F044脂肪酶N-端氨基酸序列,运用生物信息学方法,找到与黑曲霉脂肪酶基因同源的候选基因A84689。根据该基因序列,设计引物直接PCR扩增得到黑曲霉脂肪酶全长基因anl。anl全长1044bp,含3个内含子,编码297个氨基酸(含信号肽27个氨基酸),与其它脂肪酶基因没有明显同源性。将编码成熟脂肪酶的anl连接到pET28a载体上得到重组表达质粒,转化大肠杆菌BL21(De3),诱导表达并纯化出目的蛋白。通过大量稀释和DEAESepharoseFastFlow层析相结合的方法,变性后的纯化蛋白在体外实现再折叠复性。     

Cloning of a xylanase gene from Aspergillus usamii and its expression in Escherichia coli

The complete gene xyn// that encodes endo-1,4-beta-xylanase secreted by Aspergillus usamii E001 was cloned and sequenced. The coding region of the gene is separated by only one intron. It encodes 184 amino acid residues of a protein with a calculated molecular weight of 19.8kDa plus a signal peptide of 27 amino acids. The amino acid sequence of the xyn// gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL, and xylanase activity was measured. The expressed fusion protein was analyzed by SDS-PAGE and a new specific band with molecular weight of about 20kDa was found when induced by IPTG. Enzyme activity assay verified the recombinant protein as a xylanase. A maximum activity of 49.6Umg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-28a-xyn//. The xylanase had optimal activity at pH 4.6 and 50 degrees C. This is the first report on the cloning of a xylanase gene from A. usamii.     

缩短的人巨噬细胞集落刺激因子（M－CSF）在大肠杆菌中的表达

本文用聚合酶链反应（ＰＣＲ）获得了一个缩短的人巨噬细胞集落刺激因子（编码３～１４９氨基酸）ｃＤＮＡ基因,并克隆在质粒ｐＥＴ３ｄ中,在Ｔ７启动子指导下,在大肠杆菌ＢＬ２１（ＤＥ３）ＬｙｓＥ中获得了和一个６组氨酸短肽标签的融合表达。重组的融合ｍ－ＣＳＦ表达量占菌体总蛋白的１２％,表达产物一部分以不溶性包涵体形式存在,另一部分则以可溶性蛋白存在。经过金属螫合亲和层析一步纯化,所得的融合（Ｈｉｓ）６－Ｍ－ＣＳＦ在还原型ＳＤＳ－ＰＡＧＥ上基本呈一条均一的蛋白质条带。     

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Molecular Cloning of Giant Panda Pituitary Prolactin cDNA and Its Expression in Escherichia coli

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.     

Expression of cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase in Escherichia coli. Differences in kinetic mechanisms

The cDNA sequences encoding mature and precursor forms of human dihydrolipoamide dehydrogenase (E3) were expressed in Escherichia coli using a lambda PL promoter-driven prokaryotic expression vector. The expressed proteins in total cell extracts were identified by Western blot analysis using anti-pig heart E3 antibody and also by measurement of E3 activity. Most of the expressed human E3 polypeptides (five bands) were found in the insoluble pellet while primarily full-length mature E3 was found in the soluble fraction. About 2% of the total soluble protein was mature human E3 when expressed in wild type E. coli AR120. Since wild type E. coli has its own endogenous E3 activity, the expression of human E3 was performed in a pyruvate dehydrogenase complex-deficient strain of E. coli, JRG1342. The expressed recombinant human E3s in JRG1342 were purified to near homogeneity. The amino-terminal amino acid sequence analysis revealed that the recombinant mature E3 had an expected sequence while the recombinant precursor E3 lost 19 amino acid residues of its 35-amino acid leader sequence presumably due to a proteolytic cleavage. The recombinant mature E3 displayed comparable kinetic properties to those reported for highly purified mammalian E3s. The truncated precursor E3 showed about half of the mature E3 activity. The double-reciprocal plot for the mature E3 in the direction of NAD+ reduction showed parallel lines (ping-pong mechanism) while that for the truncated precursor E3 displayed intersecting lines (sequential mechanism). In the direction of NADH oxidation, the kinetic mechanisms of both E3s were apparently a ping-pong mechanism. These kinetic results showed that the partial 16-amino acid extension in the leader sequence changed the kinetic mechanism of human E3 so that it resembled that of glutathione reductase.