苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶（serine protease, SP）基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP（GenBank登录号：JX866720）,该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP（GenBank登录号：CAA72962）的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。     

华北大黑鳃金龟两种围食膜蛋白cDNA的分子克隆与序列分析

本文以华北大黑鳃金龟Holotrichia oblita中肠为材料,依据Stratagene公司文库构建试剂盒方法,构建其中肠cDNA表达文库,该文库滴度为1.9×106 pfu/mL,重组率为99.97%。依据现代免疫学原理,利用棉铃虫Helicoverpa armigera围食膜蛋白多克隆抗体筛选文库,得到两个编码华北大黑鳃金龟围食膜蛋白的cDNA克隆Ho-Peritrophin1与Ho-Peritrophin2,其cDNA长分别为2 385 bp和1 633 bp,在PolyA末端上游各有3个多聚腺苷酸信号序列AATAAA,最长开放阅读框(ORF)分别编码729个和477个氨基酸,与粉纹夜蛾Trichoplusia ni CBP2(chitin binding protein 2)的相似性最高,分别为21.9%和19.1%。结构域分析表明,Ho-Peritrophin1与Ho-Peritrophin2分别具有9个和6个几丁质结合功能域,只含有较少的O-糖基化位点,不含有类粘蛋白结构域。胰蛋白酶和胰凝乳蛋白酶对两种蛋白的作用位点主要位于几丁质结合功能域(chitin binding domain, CBD)内部,而因受几丁质结合功能域保护,这两种蛋白能够抵抗这些蛋白酶的降解。与正常CBD比较,这两种蛋白C端的CBD只含有4个Cys,只在第1与第3、第4与第5个Cys之间形成两对二硫键,缺少由第2与第6个Cys形成的二硫键。推测其N端还应包括信号肽序列和几丁质结合功能域的未知序列。     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

柑橘全爪螨酚氧化酶原激活酶基因cDNA克隆及生物信息学分析

酚氧化酶原激活酶(prophenoloxidase activating enzyme,PPAE)是酚氧化酶激活系统(prophenoloxidase activating system,PPO-AS)的组成部分,是无脊椎动物抵御入侵微生物的关键酶。本研究利用RACE技术从柑橘全爪螨Panonychus citri(McGergor)体内获得一条PPAE基因全长cDNA,命名为PcPPAE(GenBank:KC136292),属于丝氨酸蛋白酶家族。该基因cDNA全长1 676 bp,开放阅读框1 377 bp,编码458个氨基酸。该基因编码蛋白预测分子量为49.9 ku,理论等电点(pI值)为5.68,分子式为C2201H3467N607O678S22,不稳定系数为41.51,总亲水性系数为-0.268。经序列比对发现该基因具有发夹结构域,丝氨酸蛋白酶结构域,以及丝氨酸蛋白酶结构域中保守的催化三联体。系统发育分析表明该基因与二斑叶螨发夹结构丝氨酸蛋白酶基因的亲缘关系最近。本研究首次从柑橘全爪螨体内克隆获得酚氧化酶原激活酶,为后期进行柑橘全爪螨抵御微生物侵染机制研究奠定了基础。     

苦荞查尔酮合成酶基因CHS的结构及花期不同组织表达量分析

采用同源克隆、染色体步移和RT-PCR技术,首次克隆到苦荞查尔酮合酶基因（CHS）的全长DNA序列和cDNA开放阅读框（ORF)序列. 序列分析表明,苦荞CHS DNA序列（GU172165）全长1 632 bp,含1个445 bp的内含子;cDNA编码区（HM852753）全长1 188 bp,编码395个氨基酸,命名为FtCHS. 生物信息学分析表明,FtCHS和推导的氨基酸序列与其它植物CHS基因同源率在95%以上,含有CHS多基因家族的标签序列（GFGPG）、活性位点、底物结合口袋位点和环化反应口袋位点. 半定量RT-PCR分析苦荞花期FtCHS空间表达模型表明,其表达量未成熟种子＞叶＞茎＞花＞根＞成熟种子,与苦荞芦丁含量的分布基本一致,具有组织特异性.     

来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析

从一株产乳糖酶的亮白曲霉（Aspergillus candidus)中克隆到了乳糖酶基因组DNA及cDNA序列（EMBL AC－CESSION No.AJ431643),序列分析表明,乳糖酶基因组DNA序列长3458bp,其中含有8个内含子,cDNA编码区长3015bp,共编码1005个氨基酸,前19个氨基酸为信号肽序列,氨基酸序列中共含有11个潜在的糖基化位点。将此基因在不同来源的乳糖酶基因序列进行比较发现,该基因与绝大多数乳糖酶基因同源性较低。虽与米曲霉ATCC20423的乳糖酶序列同源性较高,但其在酶学性质上更优于后者,亮白曲霉的乳糖酶基因可能是一个具有更广阔的生产应用前景的新基因。     

草地螟羧肽酶A基因的克隆、表达及序列分析

羧肽酶是昆虫体内重要的消化酶系之一。利用甜菜夜蛾Spodoptera exigua（Hübner）围食膜多克隆抗体免疫筛选草地螟Loxostege sticticalis L.中肠cDNA表达文库, 得到编码羧肽酶A的全长cDNA克隆。该cDNA克隆全长1 380 bp （GenBank登录号EU924506）, 开放阅读框长1 302 bp, 编码434个氨基酸, 预测分子量和等电点分别为49.1 kDa和9.56。序列含有胰蛋白酶切割位点和催化特征, 具有典型的羧肽酶A特性。将该基因与pET30载体重组后, 经IPTG诱导, 蛋白在大肠杆菌中获得了表达。     

黄粉虫胰蛋白酶样酶基因TMTLSP全长cDNA的克隆和序列分析

以黄粉虫(Tenebrio molitor)幼虫全RNA逆转录得到的cDNA为模板,参照地鳖（Eupolyphaga sinensis）纤溶酶（fibrinolytic enzyme）简并引物,进行温度梯度PCR．以得到的扩增产物为基础,采用RACE得到基因全长cDNA,命名为黄粉虫胰蛋白酶样丝氨酸蛋白酶(Tenebrio molitor trypsin-like serine protease,TMTLSP)．TMTLSP全长869 bp(GenBank No. JN662461),开放阅读框为777 bp,编码258个氨基酸,并具有蛋白酶样特有的起始位点、活性中心预计底物结合位点．经过比对分析,该基因编码的氨基酸序列与赤拟谷盗、谷蠹、光亮扁角水虻、美洲大蠊等多种昆虫的胰蛋白酶或丝氨酸蛋白酶有较高的相似性．本研究将为胰蛋白酶样丝氨酸蛋白酶的提取及研究提供更为广泛的材料及研究依据．     

鸽源H5N1亚型禽流感病毒株的HA和NA基因特征性分析

本研究自行设计合成两对特异性引物,通过RT-PCR扩增出1株鸽源H5N1亚型禽流感病毒血凝素（HA）和神经氨酸酶（NA）两个基因的cDNA片段,将它们成功克隆于pMD18-T载体上,然后进行序列测定。结果表明,HA基因全长1707bp,编码568个氨基酸, HA基因有7个糖基化位点,在裂解位点附近有连续6个碱性氨基酸（R-R-R-K-K-R）的插入,具有高致病性毒株的分子特征。受体结合位点的氨基酸分别为YWIHELY,左侧壁氨基酸为SGVSSA,右侧壁为NGQSGR;NA基因全长1350bp,编码446个氨基酸,NA基因有3个糖基化位点。     

来源于Aspergillus candidus的乳糖酶基因的克隆及序列分析

从一株产乳糖酶的亮白曲霉(Aspergillus candidus)中克隆到了乳糖酶基因组DNA及cDNA序列(EMBL ACCESSION No. AJ431643),序列分析表明,乳糖酶基因组DNA序列长3458bp,其中含有8个内含子,cDNA编码区长3015bp,共编码1005个氨基酸,前19个氨基酸为信号肽序列,氨基酸序列中共含有11个潜在的糖基化位点。将此基因与不同来源的乳糖酶基因序列进行比较发现,该基因与绝大多数乳糖酶基因同源性较低。虽与米曲霉ATCC 20423的乳糖酶序列同源性较高,但其在酶学性质上更优于后者,亮白曲霉的乳糖酶基因可能是一个具有更广阔的生产应用前景的新基因。      

绿盲蝽丝氨酸蛋白酶基因AlSP4的克隆及取食不同寄主植物后的表达谱分析

胰蛋白酶类和胰凝乳蛋白酶类丝氨酸蛋白酶是盲蝽科昆虫消化系统内重要的消化酶。为了更好地了解丝氨酸类蛋白酶在绿盲蝽Apolygus lucorum消化系统中的作用, 本研究首次克隆了绿盲蝽丝氨酸蛋白酶基因AlSP4 (GenBank登录号为JQ609682)。序列分析结果表明, 该基因开放阅读框长999 bp, 编码332个氨基酸, 预测分子量为36.84 kDa, 理论等电点为5.35, N末端疏水区包含有16个氨基酸组成的信号肽。蛋白特征分析表明, 该基因翻译后的蛋白质具有丝氨酸蛋白酶的典型特征, 即氨基酸序列中具有组氨酸(His)、 天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件; 该基因翻译后还具有明显的胰蛋白酶前体的特征, 即此基因具有信号肽、 激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。利用荧光定量PCR技术对绿盲蝽雌、 雄成虫取食不同寄主植物后AlSP4的表达谱进行分析, 结果表明: 相对于其他寄主植物, 雌成虫取食Bt棉后AlSP4的表达量最高, 并显著高于取食常规棉后的表达量(P＜0.01)。雄成虫取食茼蒿后AlSP4的表达量最高; 雄成虫取食Bt棉后, AlSP4的表达水平仅次于取食茼蒿后的表达量, 也显著高于取食常规棉后的表达量(P＜0.01)。由此可见, AlSP4是绿盲蝽取食Bt棉后的重要消化酶基因, 对绿盲蝽适应Bt棉取食具有重要作用。     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

RNKP-1, a novel natural killer-associated serine protease gene cloned from RNK-16 cytotoxic lymphocytes

From an RNK-16 lambda-gt11 library, we have isolated and sequenced a novel cDNA rat NK cell protease 1 (RNKP-1) that has characteristics unique to serine proteases. The cDNA clone is 1102 bp and contains a complete open reading frame with an AUG start codon and a TAA stop codon. The open reading frame translates into a protein of 248 amino acids that has one glycosylation site. The characteristic N-terminal Ile-Ile-Gly-Gly and the His, Asp, and Ser amino acid residues that form the catalytic triad of serine proteases are present. The nucleotide and amino acid sequences have 87 and 80% identity, respectively, with the murine CTL-specific serine protease CCPI. However, there are extensive differences in the substrate binding regions of these proteases. Comparison of hydropathic profiles and amino acid sequences of other proteases indicate that RNKP-1 is distinct and belongs to the subfamily of serine proteases of bone marrow origin. Northern blot analysis of poly A+ RNA from rat splenocytes cultured with Con A showed 1000 and 1400 nucleotide mRNA are detected with RNKP-1 after 1 day of Con A-stimulation. The expression of the two mRNA bands continues through day 5 of culture with the lectin and may represent RNKP-1 mRNA plus related sequences due to cross-hybridization. RNKP-1 is also expressed in RNK-16 cells, but is not expressed in freshly isolated rat splenocytes, brain, lung, or lymph node tissues. The induction of RNKP-1 expression in the Con A-cultured spleen cells is accompanied by increases in both NK and lymphokine-activated killer lymphocyte activities. These data indicate that RNKP-1 is a unique serine protease that may be preferentially expressed in NK cells.     

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Cloning of the cDNA for the serine protease homolog CAP37/azurocidin, a microbicidal and chemotactic protein from human granulocytes

Human cationic antimicrobial protein (CAP37) is a neutrophil granule protein with monocyte chemotactic and antibacterial activity. A CAP37 cDNA clone of 899 bp was isolated from an HL-60 cDNA library using degenerate oligonucleotide probes based on partial N-terminal sequence of the CAP37 protein. The cDNA sequence predicts an open reading frame of 753 bp encoding a protein of 251 amino acids. A 26-residue eukaryotic signal peptide and a potential 7 amino acid pro-peptide are present at the N-terminus of the protein. The cDNA sequence also predicts three N-linked glycosylation attachment sites and eight intramolecular cysteines. The deduced amino acid sequence of CAP37 shows 44, 42, and 32% homology at the amino acid level to neutrophil elastase, myeloblastin, and cathepsin G, respectively, suggesting that CAP37 is a member of the serine protease gene family. CAP37 does not possess serine protease activity probably due to mutations in two of three residues in the catalytic triad of the "charge relay system." Whereas CAP37 is expressed in undifferentiated HL-60 cells no message is detected in mature neutrophils.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.     

A novel serine protease with clip domain from scallop Chlamys farreri

The serine proteases with clip domain are involved in various innate immune functions in invertebrate such as antimicrobial activity, cell adhesion, pattern recognition and regulation of the prophenoloxidase system. A serine protease with clip-domain cDNA (Cf SP) was obtained by Expressed sequence taggings (ESTs) method and rapid amplification of cDNA ends (RACE). The Cf SP full-length cDNA was of 1,152 bp, including a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 81 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 1,008 bp encoding a polypeptide of 336 amino acids with a putative signal peptide of 19 amino acids. The deduced amino acid sequence of Cf SP contained an amino-terminal clip domain with three disulfide bonds formed six conserved Cys residues, a carboxyl-terminal trypsin-like domain with the conserved His-Asp-Ser catalytic triad, and a low complexity linker sequence. The Cf SP was strongly expressed in hemocytes and the mRNA expression of Cf SP was up-regulated and increased 3.2-fold and 2.6-fold at 16 h after injection of Vibrio anguillarum and Micrococcus luteus. The results suggested that Cf SP gene might be involved in immune response of Gram-negative and Gram-positive microbial infection in scallop.