棉铃虫核多角体病毒iap3基因表达时相分析

目的:原核表达棉铃虫核多角体病毒(Helicoverpa armigera nucleopolyhedrovirus,HearNPV)iap3基因,制备该蛋白的多克隆抗体,并利用该抗体分析iap3基因在病毒感染过程的表达时相,为深入研究提供基础.方法:PCR扩增iap3基因后,克隆至pET28b,转化到大肠杆菌BL21 (DE3)中诱导表达,利用亲合层析进行蛋白纯化,将纯化融合蛋白免疫大鼠制备抗血清,利用抗血清Western blot检测IAP3在病毒感染过程的表达时相.结果:成功在原核细胞中表达iap3基因,并获得纯化的融合His - tag的IAP3蛋白,制备了该蛋白的多克隆抗体.发现iap3基因最早在感染后24h表达,到72h到达表达高峰.结论:获得了IAP3多克隆抗体,iaP3基因是一个晚期表达基因.     

人类博卡病毒(HBoV)非结构蛋白NS1基因的克隆、原核表达及多克隆抗体的制备

目的:克隆、表达、纯化人类博卡病毒(HBoV)非结构蛋白NS1,制备抗NS1多克隆抗体。方法:利用PCR扩增HBoV非结构蛋白NS1基因,将其克隆至pMAL-c2X表达载体上,重组质粒转化大肠杆菌DH10B,IPTG诱导表达。表达的融合蛋白经Amylose Resin亲和层析柱纯化后,免疫新西兰大白兔制备多克隆抗体。用间接ELISA法检测抗体效价。结果:原核表达融合蛋白MBP-NS1,并获得了其多克隆抗体,抗体效价达到1∶32000。结论:在原核表达系统中表达、纯化了融合蛋白,制备抗NS1多克隆抗体,为进一步研究该病毒非结构蛋白基因的转录和翻译机制提供可靠的工具。     

弓形虫MIC2胞质尾段蛋白及其多克隆抗体的制备

目的:制备弓形虫微线体蛋白2胞质尾段(MIC2C)蛋白片段及其多克隆抗体。方法:以弓形虫cDNA文库为模板,PCR扩增135bp MIC2C基因片段,构建MIC2C/pGEX-4T-1原核表达系统;IPTG诱导表达GST-MIC2C融合蛋白;用纯化的融合蛋白加免疫佐剂背部皮内注射免疫新西兰兔,制备多克隆抗体,亲和层析纯化并分析抗体的效价。结果:构建了MIC2C原核表达系统,表达并纯化了GST-MIC2C融合蛋白,蛋白的浓度为3.07mg/ml;获得了抗该蛋白的兔源性抗血清,纯化后的多克隆抗体效价为1:16 000。结论:在体外制备并纯化了GST-MIC2C融合蛋白及其多克隆抗体,为后续弓形虫入侵机制的研究奠定了基础。     

大鼠Scratch2蛋白锌指结构域的表达与纯化

[目的]原核表达大鼠Scratch2基因的C_2H_2型锌指结构域,纯化获得GST-C_2H_2融合蛋白。[方法]以新鲜大鼠前额叶脑组织cDNA为模板,利用PCR扩增带有EcoRⅠ和XhoⅠ酶切位点的Scratch2基因的锌指结构域后,构建原核表达载体pGEX-4T-1-C_2H_2,并将其转化至大肠杆菌BL21(DE3),经IPTG诱导融合蛋白表达,再利用MagneGST particles亲和纯化GST-C_2H_2融合蛋白,最后通过Western Blot鉴定此融合蛋白。[结果]成功构建了pGEX-4T-1-C_2H_2原核表达载体;在20℃、0.2 mmol/L的IPTG诱导下,GST-C_2H_2融合蛋白即可有效地表达;经MagneGST particle纯化的GST-C_2H_2蛋白可被识别Scratch2锌指结构域的抗体所识别。[结论]纯化的GST-C_2H_2蛋白可用于后续研究。     

水稻YTB中OSVDAC5蛋白的原核表达与抗体制备

目的:克隆水稻YTB osvdac5基因,原核表达后获得纯化的OSVDAC5蛋白,制备相应的抗体.方法:采用Trizol法提取水稻总mRNA,反转录为cDNA,通过PCR扩增得到该基因与原核表达载体连接,构建重组质粒pET-30a-osvdac5,并转入大肠杆菌进行原核表达,SDS-PAGE检测表达产物.通过镍柱纯化获得的单一目的蛋白用于抗体制备,用Western Blot检测抗体的特异性.结果:克隆到原核表达载体中osvdac5基因的ORF为813 bp,编码271个氨基酸.在大肠杆菌中15℃、0.7mmol/L的IPTG浓度诱导17 h是pET-30a-osvdac5融合蛋白表达的优选条件,表达的OSVDAC5蛋白属于包涵体蛋白.镍柱纯化后的OSVDAC5为30 kD左右的单一条带.Western Blot分析表明,抗体能够与30 kD处的OSVDAC5蛋白进行特异性结合.结论:成功克隆了水稻YTB osvdac5基因,原核表达蛋白OSVDAC5制备的多免隆抗体具有一定特异性,能与免疫抗原结合,这为进一步研究OSVDAC5蛋白在植物不同生长发育时期中的表达模式奠定了基础.     

沙门菌毒力基因spvB的原核表达及其多克隆抗体的制备

目的:构建沙门菌毒力基因spvB的原核表达载体,诱导表达纯化SpvB蛋白并以其为抗原免疫小鼠,制备多克隆抗体。方法:利用生物信息学软件对SpvB进行分析,选取抗原性较高、易表达的氨基酸序列作为克隆序列,以携带spvB基因的鼠伤寒沙门菌为模板,PCR扩增目的片段后与原核表达载体pET28a(+)连接;将质粒pET28a-SpvB转化大肠埃希菌BL21(DE3)后诱导表达并纯化。目的蛋白免疫小鼠,制备抗SpvB多克隆抗体,Western blot检测抗体特异性。结果:成功构建spvB原核表达载体,经IPTG诱导结果显示,重组蛋白表达且主要存在于包涵体中,将纯化后的蛋白免疫小鼠Western blot检测血清中抗体与SpvB特异性结合。结论:获得具有免疫原性的SpvB蛋白及其多克隆抗体,为进一步研究该基因的功能奠定基础。     

花生AhAO1蛋白的原核表达、纯化及抗体制备

采用RT-PCR方法,以花生叶片RNA逆转录得到的cDNA为模板,扩增出AhAO1基因片段,将其插入原核表达载体pPROEXHTa中,构建AhAO1基因片段原核表达载体HTaAhAO1,经PCR和测序确证后,以IPTG诱导其在E.coli BL21中高表达His-AhAO1融合蛋白,采用亲和层析柱与电泳纯化融合蛋白。用纯化的His-AhAO1融合蛋白制备抗体,为分析AhAO1的功能奠定基础。     

小鼠OAZ2基因的原核表达及抗体制备

目的:克隆小鼠鸟氨酸脱羧酶抗酶2(OAZ2)功能基因,原核表达、纯化OAZ2蛋白并制备抗OAZ2多克隆抗体.方法:IRT-PCR法从鼠黑色素瘤细胞总RNA中克隆OAZ2 cDNA后,通过重叠延伸PCR技术构建无需移码即可全长翻译的功能基因.将OAZ2功能基因克隆人原核表达载体pET15b并原核表达.表达的蛋白经Ni-NTA亲和层析纯化后,用SDS-PAGE和Western Blot分析鉴定.用纯化的OAZ2蛋白作为抗原免疫Bab/C小鼠以制备多克隆抗体,制备抗体用ELISA和Western Blot检测抗体滴度和特异性.结果:成功获得小鼠OAZ2 cDNA并构建出无需移码翻译的OAZ2功能基因.OAZ2功能基因在大肠杆菌BL21(DE3)中可诱导性高表达并能用Ni-NTA树脂高效纯化.用纯化蛋白免疫Bab/C小鼠制备的抗血清经ELISA检测有较高的多克隆抗体效价(＞1∶64000),经Western blot鉴定可与纯化的OAZ2蛋白质特异性结合.结论:建立了鼠OAZ2蛋白原核表达和纯化技术,制备出高效价和特异性抗OAZ2多克隆抗体,为进一步研究OAZ2基因的功能奠定了基础.     

小鼠Prune蛋白DHH结构域的原核表达及多克隆抗体的制备

[目的]表达、纯化小鼠Prune蛋白DHH结构域(m-Prune D),并制备多克隆抗体。[方法]生物信息学方法分析m-Prune D氨基酸序列;PCR扩增目的基因m-Prune D,克隆入原核表达载体p ET28a(+);IPTG诱导目的基因表达,SDS-PAGE和Western Blot鉴定蛋白表达,亲和层析法纯化蛋白;用纯化的重组m-Prune D免疫小鼠制备多克隆抗体;Western Blot检测多克隆抗体特异性。[结果]PCR成功扩增m-Prune D基因,双酶切及测序结果表明成功构建m-Prune D原核表达载体,SDS-PAGE和Western Blot鉴定表明成功表达约25 k Da的重组蛋白。纯化蛋白免疫小鼠后抗体滴度最高可达1∶25 600,所制备的多克隆抗体可特异性识别原核和真核细胞中DHH结构域蛋白。[结论]在E.coli中成功表达小鼠Prune蛋白DHH结构域,制备了多克隆抗体血清,可用于Prune蛋白生物学功能的进一步研究。     

利用GST融合蛋白表达系统表达与纯化人N-Ras蛋白

目的:克隆人N-ras蛋白全长编码区基因,获得其原核表达产物,并对融合蛋白进行纯化。方法:采用PCR技术从人乳腺文库中扩增出人N-ras蛋白全长编码区基因,将其克隆到p GEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,SDS-PAGE鉴定表达与纯化产物。结果:从人乳腺文库中扩增获得约600 bp的DNA片段,并克隆至p GEX-KG载体上,经测序与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量约47×103的目的蛋白;纯化后,经鉴定获得了纯度较高的重组蛋白GST-N-Ras。结论:获得了重组蛋白GST-N-ras,为后续深入研究Ras基因与其他癌基因、抑癌基因的相互作用奠定了基础。     

Avian SERPINB11 gene: characteristics, tissue-specific expression, and regulation of expression by estrogen

Serpins, a group of proteins with similar structural and functional properties, were first identified based on their unique mechanism of action: their inhibition of proteases. While most serpins have inhibitory roles, certain serpins are not involved in canonical proteolytic cascades but perform diverse functions including storage of ovalbumin in egg white, transport of hormones (thyroxine- and cortisol-binding globulin), and suppression of tumors. Of these, serpin peptidase inhibitor, clade B, member 11 (SERPINB11) is not an inhibitor of known proteases in humans and mice, and its function is unknown. In the present study, the SERPINB11 gene was cloned, and its expression profile was analyzed in various tissues from chickens. The chicken SERPINB11 gene has an open reading frame of 1346 nucleotides that encode a protein of 388 amino acids that has moderate homology (38.8%-42.3%) to mammalian SERPINB11 proteins. Importantly, SERPINB11 mRNA is most abundant in the chicken oviduct, specifically luminal and glandular epithelia, but it was not detected in any other chicken tissues of either sex. We then determined effects of diethylstilbestrol (DES; a synthetic nonsteroidal estrogen) on SERPINB11 expression in the chicken oviduct. Treatment of young chicks with DES induced SERPINB11 mRNA and protein only in luminal and glandular epithelial cells of the oviduct. Collectively, these results indicate that the novel estrogen-induced SERPINB11 gene is expressed only in epithelial cells of the chicken oviduct and implicate SERPINB11 in regulation of oviduct development and differentiated functions.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.     

Expression and purification of glutamine synthetase cloned from Bacteroides fragilis

A glutamine synthetase (GS) gene, glnA, from Bacteroides fragilis was cloned on a recombinant plasmid pJS139 which enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. DNA homology was not detected between the B. fragilis glnA gene and the E. coli glnA gene. The cloned B fragilis glnA gene was expressed from its own promoter and was subject to nitrogen repression in E. coli, but it was not able to activate histidase activity in an E. coli glnA ntrB ntrC deletion mutant containing the Klebsiella aerogenes hut operon. The GS produced by pJS139 in E. coli was purified; it had an apparent subunit Mr of approximately 75,000, which is larger than that of any other known bacterial GS. There was very slight antigenic cross-reactivity between antibodies to the purified cloned B. fragilis GS and the GS subunit of wild-type E. coli.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Genetic analysis of Escherichia coli O111:B4, a strain of medical and biochemical interest.

Procedures have been worked out which allow, for the first time, the genetic analysis of Escherichia coli O111:K58:H2 (O111:B4). The approximate map position of mutant loci was determined by mating with 15 Hfr strains of E. coli K-12. In addition, P1 transduction procedures were used for establishing relative gene order and linkage for any region of the E. coli O111:B4 chromosome. To obtain these, it was necessary to select for a rare P1 lysogen since E. coli O111:B4 is resistant to phage P1. Finally, genetic homology between E. coli strains K-12 and O111:B4 is suggested since they can form stable haploid hybrids, and several loci have similar map positions in the two strains.     

Synthesis and expression in Escherichia coli of the gene for the albumin-binding domain of Streptococcus protein G]

The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Bacteriophage T4 Inhibits Colicin E2-Induced Degradation of Escherichia coli Deoxyribonucleic Acid I. Protein Synthesis-Dependent Inhibition

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.     

Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma

SERPINB1 (serine protease inhibitor, clade B, member1) is a member of the SERPINB family. Recent studies suggested that SERPINB1 may suppress the migration and invasion of lung and breast cancers. In this study, we investigated a possible involvement of SERPINB1 in the regulation of hepatocellular carcinoma metastasis (HCC). The expression of SERPINB1 was evaluated using western blot analysis in 8 paired fresh HCC specimens and immunohistochemistrical assay on 67 paraffin-embedded HCC slices. SERPINB1 was downregulated in HCC specimens and correlatively related with two clinicopathologic features of HCC, metastasis (P = 0.000) and vein invasion (P = 0.006). Univariate and multivariate survival analyses showed a lower level of SERPINB1 expression is associated with poor prognosis and clinical outcome (P = 0.001). In addition, small interfering RNA targeting SERPINB1 was used to knock down the expression of SERPINB1 in Huh7 and BEL-7404 cells. We showed that interference of SERPINB1 promoted migration and invasion of HCC cells, while cell proliferation was not affected. Finally, we observed an apparent increase in the level of active matrix metalloproteinase-2 (MMP2) after SERPINB1 knockdown, implying that SERPINB1 might participate in the regulation of HCC metastasis through modulating the activation of matrix metalloproteinases. Overall, our results suggested an inhibitory role of SERPINB1 in the migration and invasion of HCC, implying that SERPINB1 might be a potential prognostic indicator of HCC metastasis.