烟曲霉几丁质酶基因的克隆与表达

Chi4 4是烟曲霉 (Aspergillusfumigatus)YJ-407产生的一种胞外几丁质酶。通过用真菌几丁质酶保守氨基酸序列与Chi44的N-端序列检索烟曲霉部分基因组序列数据库 ,获得一个编号为contig555的烟曲霉基因组序列 ,可能包含烟曲霉几丁质酶的基因。根据检索结果用RT-PCR方法从烟曲霉YJ-407中克隆到1.4kb的cDNA片段 ,该cDNA的ORF编码一个395个氨基酸的蛋白 ,分子量为43.6kD。对其推导氨基酸序列分析表明该蛋白与其它真菌来源的几丁质酶同源 ,而且活性中心与人巨噬细胞几丁质酶高度同源。该cDNA已在E .coli与PichiapastorisGS115中获得表达 ,分别获得 43kD和44kD的重组蛋白 ,两种重组蛋白均有几丁质酶活性。与野生酶相比 ,大肠杆菌表达的43kD重组酶及Pichia酵母表达的44kD重组酶稳定性下降 ,说明Chi44的糖基化修饰可稳定酶蛋白.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

酵母豆蔻酰辅酶A：蛋白质N端转酰基酶基因的克隆与表达

利用ＰＣＲ技术,从酵母染色体中扩增得到酵母豆蔻酰－ＣｏＡ：蛋白质Ｎ端转酰基酶（ＹＳＣＮＭＴ）基因,并克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ＋载体中。由ＤＮＡ全序测定表明,获得了ＹＳＣＮＭＴ编码基因。进一步构建了Ｔ７Ｐｒｏｍｏｔｅｒ控制下的含上述完整ＹＳＣＮＭＴ编码基因的表达质粒ｐＭＦＴ７－５－ＮＭＴ,转化大肠杆菌ＢＬ２１（ＤＥ３）,进行ＩＰＴＧ诱导表达研究。通过ＳＤＳ－ＰＡＧＥ分析,观察到一与理论分子量一致的诱导条带（约５３ｋＤ）,占全菌蛋白的３９％左右,且可溶性部分约占上清液中全部蛋白的３４％。经一步Ｐ１１磷酸纤维素阳离子交换柱层析,将其纯化到纯度达９７％以上．纯化的表达产物经Ｎ端氨基酸序列分析,所测定的Ｎ端５个氨基酸的序列,与从克隆的ＹＳＣＮＭＴ基因推出的氨基酸序列完全一致（不含Ｎ端Ｍｅｔ）。对所得的ＹＳＣＮＭＴ进行酶活力鉴定,观察到了明显的活力。     

人巨细胞病毒结构蛋白p150、p38的原核表达纯化及免疫性鉴定

应用聚合酶链式反应(PCR)扩增HCMV结构蛋白p150aa595~aa614/aa1006~aa1048和p38aa117~aa373基因片段,将其分别克隆于表达载体pGEX-4T-1和pET30 a-SetB中,转化大肠杆菌BL21(DE3),IPTG诱导表达,重组质粒经酶切鉴定及DNA测序证实pGEX-4T-1/p150aa595~aa614/aa1006~aa1048和pET30 a-SetB/p38aa117~aa373构建正确;通过优化表达和纯化,分别获得分子量约为35kD和43kD纯化的表达产物。经亲和层析纯化以免疫印迹实验对纯化的重组蛋白进行免疫反应性检测,证实重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373能够与抗HCMV IgM阳性血清反应,说明表达的重组蛋白p150aa595~aa614/aa1006~aa1048、p38aa117~aa373具有良好的免疫性,对HCMV的原发感染具有潜在的应用价值。     

人骨形成蛋白2A活性片段在大肠杆菌中的高效表达

将编码人骨形成蛋白２Ａ（ＢＭＰ２Ａ）Ｃ端１７３个氨基酸（ＢＭＰ２３）和１３４个氨基酸（ＢＭＰ２４）的ＤＮＡ基因片段分别重组克隆进入ＰＬ启动子控制下的表达载体,构建了表达质粒ｐＢＬＢＭＰ２３和ｐＢＬＢＭＰ２４,分别转化大肠杆菌进行表达研究．ＳＤＳ－ＰＡＧＥ分析温敏诱导的表达菌,可以分别观察到分子量为２０ｋＤ和１５．５ｋＤ的高表达条带,与理论计算的分子量一致,表达量分别占细菌蛋白质总量的１０％和２０％左右。表达产物经包含体制备达到８０％以上纯度。Ｎ端序列测定的１５个氨基酸,与重组ｃＤＮＡ基因编码的序列相同．ＢＭＰ２３和ＢＭＰ２４包含体经复性处理后,得到二聚体分子蛋白质条带,与骨基质胶原重组后在大鼠体内测活,观察到ＢＭＰ２３诱导软骨细胞生成,ＢＭＰ２４刺激丰富的骨样胶原组织合成．     

Ataxin-3融合蛋白表达、纯化及抗血清的制备

目的:表达GST-ataxin-3-N融合蛋白并制备GST-ataxin-3特异性抗体,为深入研究其功能及其在SCA3发病机制中的作用提供重要的技术和材料保障.方法:将人ataxin-3氨基端基因克隆入原核表达载体pGEX-4T-2,在大肠杆菌(E.coli)BL21中表达,用Glutathione sepharose4B凝胶亲和柱纯化目的蛋白.利用纯化的GST-ataxin-3-N蛋白制备多克隆抗体.结果:成功构建了原核表达载体,得到高表达量的融合蛋白,经亲和层析柱纯化获得较高纯度的GST-ataxin-3-N融合蛋白.以融合蛋白免疫新西兰兔得到Ataxin-3-N多克隆抗体,Western Blotting及免疫荧光均证实该抗体能够识别Ataxin-3-myc蛋白,具有较高特异性.结论:利用原核表达人GST-ataxin-3-N融合蛋白制备的Ataxin-3多克隆抗体具有较好的特异性,可用于该蛋白的相关研究.     

红斑丹毒丝菌SpaA蛋白保护区域的免疫学检测

为了确定菌体表面保护性抗原A（SpaA）的免疫保护区域,通过PCR从红斑丹毒丝菌C43065株基因组DNA中分别扩增出编码成熟SpaA及其N端342个氨基酸序列（SpaA-N）和C端160个氨基酸序列（SpaA—C）的基因片段,将它们分别克隆到表达载体pET32a上,转化入大肠埃希菌BL21,IPTG诱导,亲和层析法纯化重组蛋白,并检测它们对小鼠的保护作用。SDS—PAGE结果显示重组SpaA、SpaA-N和SpaA—C以可溶性形式表达在大肠埃希菌BL21细胞质中并具有良好的免疫原性。保护试验结果表明重组SpaA、SpaA—N和NaOH提取抗原完全保护小鼠受强毒株C43065的致死性感染,但重组SpaA—C没有保护作用。结果表明SpaA-N可以作为预防猪丹毒的亚单位疫苗。     

Proteolysis of ribosomal protein S1 from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus thermophilus</Emphasis> leads to formation of two different fragments

As a result of limited tryptic proteolysis of S1 ribosomal protein (molecular mass 60 kD) from Thermus thermophilus, 25 N-terminal amino acid residues and 71 C-terminal amino acid residues are split off and a stable high-molecular-weight fragment with molecular mass of 49 kD is formed that retains RNA-binding properties and is capable of interacting with 30S ribosomal subunit. Earlier, application of a similar procedure for the formation of a fragment of S1 protein from Escherichia coli resulted in splitting of 171 N-terminal amino acid residues with the formation of a 41.3 kD fragment that possesses RNA-binding properties only. Thus, in spite of high homology between E. coli and T. thermophilus proteins, the proteolysis leads to the formation of two different fragments, which points, in our opinion, to the fact of significant differences between their structures.     

Clostridium botulinum types A,B, C1, and E produce proteins with or without hemagglutinating activity: Do they share common amino acid sequences and genes?

Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C₁, and E) and simultaneously proteins, A Hn⁺, B Hn⁺, C Hn⁺, and E Hn^?, that have high, low, and no hemagglutinating activity. A Hn⁺ and B Hn⁺ are serologically cross-reactive. A Hn⁺, B Hn⁺, and C Hn⁺ found as large aggregates (900–220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn⁺, B Hn⁺ is 17 kD and 27 kD for C Hn⁺. The 116 kD E Hn^? does not aggregate. We determined the sequences of 10–33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn⁺ and B Hn⁺. Each of these subunits have unique sequences, indicating that the larger units studied are not homomers or heteromers of smaller units. The subunits of A Hn⁺ and B Hn⁺ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity).In vitro proteolysis of 116 kD E Hn^? with different proteases did not impart hemagglutinating activity to the fragments. The 116 kD E Hn^? and one of its proteolytic fragments (87 kD) were partially sequenced. Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn⁺, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzukiet al., 1990). The derived amino acid sequence of this open reading frame, we found, has 73–84% sequence identity with those of the 17 kD subunits of A Hn⁺ and B Hn⁺ and significant identity with the N-terminal of E Hn^?. These highly conserved sequences show existence of genetic linkage among the Hn⁺ and Hn^? proteins.     

大麦黄矮病毒GAV株系外壳蛋白基因在大肠杆菌中的高效表达

大麦黄矮病毒 (ＢＹＤＶｓ)是一种由蚜虫专化性传播的循回非增殖型单链正义ＲＮＡ病毒 ,是黄症病毒属 (Ｌｕｔｅｏｖｉｒｕｓ)的代表成员。ＢＹＤＶＧＡＶ是我国的主流株系之一 ,它与美国的ＢＹＤＶＭＡＶ有较强的血清学关系 ,但美国的ＭＡＶ仅由麦长管蚜传播 ,而我国的ＧＡＶ除麦长管蚜传播外 ,还可由麦二叉蚜传播[1,2 ] 。近年来 ,有关麦蚜传播病毒专化性的研究已成为ＢＹＤＶ的研究热点[3 ] 。ＢＹＤＶ病毒的传播涉及病毒粒子表面成分与蚜虫体内专化性受体的相互作用 ,ＢＹＤＶ病毒粒子表面主要成分是ＣＰ ,另外一个次要成分是通读蛋白 (…     

CMY-39型AmpC酶新基因亚型的序列分析与原核表达

目的对弗劳地枸橼酸杆菌所产CMY-39型AmpC酶新基因亚型进行基因克隆和重组表达。方法以产CMY-39型AmpC酶新基因亚型的弗劳地枸橼酸杆菌总基因组DNA为模板,PCR扩增CMY-39,将其克隆入pGEM-T载体后测定该核苷酸序列,再将CMY-39基因克隆到pET-32 a（＋）系统进行重组,重组菌在大肠埃希菌BL21中表达,SDS-PAGE电泳鉴定酶蛋白的表达。结果 PCR扩增出大小为1 146 bp的基因片段,与GenBank上CMY-39的基因序列同源性为99%。大肠埃希菌BL21转化pET-32 a（＋）/CMY-39重组质粒后,AmpC酶三维试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示,蛋白分子质量大约为60 kD。结论此基因为CMY-39新基因亚型,登陆号为HM565135;成功表达重组的CMY-39型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定了基础。     

麻疯树核糖体失活蛋白基因的克隆和表达

麻疯树(Jatropha curcas L.)核糖体失活蛋白(curcin)是存在于麻疯树种子中的一种毒性较强的蛋白,它与蓖麻毒蛋白和相思子毒蛋白的性质相似,属Ⅰ型核糖体失活蛋白.从麻疯树种子中分离得到一种分子量为28.2 kD的蛋白质,其对无细胞系统中蛋白质合成的抑制活性较强,IC50为(0.19±0.01)nmol/L,具有RNA N-糖苷酶活性.依据curcin的N端部分氨基酸设计简并引物,通过RT-PCR和5'-RACE技术从未成熟种子总RNA中克隆到curcin全长cDNA序列.该cDNA全长由1 173个碱基组成,包含一个编码293个氨基酸的前体蛋白,前42个氨基酸为信号肽.推测的多肽序列与测定的蛋白质N端序列相同,与多种己发表的Ⅰ型核糖体失活蛋白和Ⅱ型核糖体失活蛋白的A链有一定的同源性.将curcin的编码区与表达载体pQE-30相连后,转入大肠杆菌(Escherichia coil)M15菌株中得到了有效的表达.将表达的融合蛋白纯化后发现,它具有抑制无细胞系统蛋白质合成的能力.     

Characterization of adhesive epitopes with the OmpS display system.

OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins. Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed. According to this model, OmpS contains 18 transmembrane beta-strands and nine surface-accessible loops. Adhesive epitopes can, when inserted into surface-accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics. We inserted three D-repeats from the Staphylococcus aureus fibronectin-binding protein FnBPA into L4 of OmpS and showed that E. coli cells expressing these hybrids bind fibronectin. DNA fragments covering the N-terminal half of the globoside-binding P-fimbrial adhesin class II PapG of E. coli were cloned into the same surface accessible loop (L4) of OmpS. Fragments of papG encoding 53 or 186 amino acids from the N-terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside. Removal of 23 amino acids from the N-terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it. The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins. We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG. This antiserum recognized the P-fimbriae on E. coli cells. These data provide evidence that the N-terminal first 53 amino acids of class II PapG contain the receptor-binding domain.     

Human neutrophil response to recombinant neisserial Opa proteins

Interactions of human neutrophils with recombinant Escherichia coli expressing gonococcal outer membrane Opa proteins were examined using chemiluminescent and biological assays. Seven opa loci from Neisseria gonorrhoeae MS11 4.8 were expressed as beta-lactamase-Opa fusion proteins that contained all but the mature N-terminal amino acid of the full-length Opa protein fused to three N-terminal amino acids derived from the mature beta-lactamase. The Opa fusion proteins were exported and assembled in the outer membrane of E. coli in a manner similar to that of Opa in N. gonorrhoeae, as evaluated by antibody binding and in situ proteolytic cleavage. All fusion proteins exhibited the characteristic heat-modifiable migration in SDS-polyacrylamide gel electrophoresis that typifies Opa proteins of neisseriae. Opa fusion proteins conferred on E. coli the ability to stimulate a chemiluminescent response from human neutrophils in the absence of antibody or complement. The nature of the response in terms of chemiluminescence, phagocytosis, and killing was in all cases analogous to that seen using N. gonorrhoeae expressing the equivalent Opa protein. Neither E. coli nor gonococci expressing OpaA elicited a response from neutrophils. Use of E. coli expressing Opa fusions should be useful in defining their biological activities and pathogenic roles.     

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.     

Codon optimization of MTS1 and its expression in Escherichia coli

MTS1, which encodes a protein named p16, is an important gene involved in tumorigenesis. To increase the expression of p16 in Escherichia coli, MTS1 was synthesized de novo by recursive PCR, with codons optimized towards E. coli. Studies indicate that N-terminal amino acids of p16 had negative impact on its expression in E. coli. The function of p16DeltaN8 is not affected by the absence of N-terminal eight amino acids, compared with p16. p16DeltaN8 was expressed in E. coli, which reached 22% of total cell proteins. Purified p16DeltaN8 (purity was 98%) was delivered into A875 (melanoma), MCF7 (breast cancer), and HeLa (cervical cancer) cells by lipofectin. Results show purified p16DeltaN8 remarkably inhibited the growth of A875 and MCF7 cells, whereas it had little effect on HeLa cells.     

High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli

We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.     

Genes encoding alpha-heavy chains of rabbit IgA: characterization of cDNA encoding IgA-g subclass alpha-chains

cDNA molecules encoding rabbit IgA alpha-heavy chains have been synthesized and six of these have been characterized. The complete nucleotide sequence of one cDNA, p 19 (942bp), showed that it encoded all but the N-terminal 57 amino acid residues of the constant region of alpha-chains. The cDNA molecules were subcloned into the expression vector pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that all six cDNA molecules encoded alpha-chains of the IgA-g subclass. Comparison of the amino acids encoded by the alpha-cDNA with the amino acid sequence of mouse and human alpha-chains showed that although all of the intradomain disulfide bonds appear to be conserved, some positions, probably involved in interchain disulfide bonds, are not conserved. We propose that secretory component is covalently bound to cysteine 299 and/or cysteine 301 of the CH2 domain of mouse and human alpha-chains. The results from Southern blot analysis of genomic DNA with 32P-cDNA suggests that the rabbit genome has multiple C alpha genes.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.