猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S.suis 2)05ZYH33全基因序列进行生物信息学分析,并与相关家族蛋白进行同源性比较,设计合成引物,PCR法扩增出约1.3 kb的烯醇化酶编码基因(enolase,eno),将其克隆入pMD-18T载体中,进一步亚克隆入表达载体pET32a.将重组表达质粒pET32a::eno转化E.coli BL21(DE3),经IPTG诱导表达后,SDS-PAGE初步检测到分子量约为75kD的蛋白带.通过His-Tag亲和层析纯化,获得融合蛋白His-ENO.Western-blot表明该表达产物具有免疫原性.基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S.suis 2 05ZYH33细菌的表面.这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用.     

猪链球菌2型烯醇化酶的分子克隆与免疫学特性

对新近测定的猪链球菌2型(S. suis 2) 05ZYH33全基因序列进行生物信息学分析, 并与相关家族蛋白进行同源性比较, 设计合成引物, PCR法扩增出约1.3 kb的烯醇化酶编码基因 (enolase, eno), 将其克隆入pMD-18T载体中, 进一步亚克隆入表达载体pET32a。将重组表达质粒pET32a::eno转化E. coli BL21 (DE3), 经IPTG诱导表达后, SDS-PAGE初步检测到分子量约为75kD的蛋白带。通过His-Tag亲和层析纯化, 获得融合蛋白His-ENO。Western-blot表明该表达产物具有免疫原性。基于ELISA进行的细胞定位实验证实了Enolase可以部分存在S. suis 2 05ZYH33细菌的表面。这提示了Enolase作为一种新发现的抗原对于引发猪链球菌相关疾病可能发挥着重要的作用。     

马链球菌透明质酸合成酶基因的分子克隆及表达

根据Streptococcus equisimilis、Streptococcus pyogenes、Streptococcus uberis三种链球菌透明质酸合成酶(seHAS、spHAS、suHAS)基因的高度保守区,设计一对简并引物,用两次PCR从Streptococcus equi的总DNA中扩增出sqHAS基因。构建表达质粒pSE-sqHAS并转化大肠杆菌DH5α,诱导培养后在细胞膜中检测到sqHAS蛋白及活性。利用携带该酶的细胞膜以UDP-GlcA和UDP—GlcNAc为底物在体外合成了分子量为3.6×10~6Da的HA,分别是发酵法生产和提取法生产的HA的分子量的2.5倍和5倍左右。马链球菌透明质酸合成酶基因的克隆及表达,国内外文献尚未见报道。本研究为体外酶法生产透明质酸做了初步探索。     

2型猪链球菌srtBCD菌毛岛SSU2100基因的克隆表达与免疫学特性

【目的】研究2型猪链球菌(Streptococcus suis serotype 2,S.suis 2)野毒株05ZYH33的srtBCD菌毛岛菌毛亚蛋白SSU2100的免疫保护性作用。【方法】通过PCR扩增出SSU2100基因片段,将目的基因克隆到表达载体pET28a上,转化入E.coli BL21感受态中表达,亲和层析法纯化目的蛋白;Western blot检测SSU2100蛋白的免疫原性,重组蛋白免疫BALB/c小鼠,ELISA法检测多抗血清的效价及IgG亚型,研究重组蛋白的免疫保护作用。【结果】在原核系统成功表达出了SSU2100蛋白;ELISA结果显示重组蛋白能够刺激小鼠产生高效价的免疫抗体;动物实验表明该蛋白具有良好的免疫保护作用。【结论】菌毛亚蛋白SSU2100可以作为S.suis 2亚单位疫苗的候选分子,为系统地阐释srtBCD菌毛岛在S.suis 2致病机制中的作用奠定基础。     

Molecular cloning and characterization of soybean peroxidase gene families

Plant peroxidases play major roles in many physiological processes. A soybean seedbud (21 days after flowering) Uni-ZAP XR cDNA library was screened with a peroxidase-specific probe. The probe was generated by 3′ rapid amplification of cDNA ends with soybean seedbud total RNA and a degenerate primer derived from a plant peroxidase conserved amino acid region (distal heme ligand). Positive clones were recovered by PCR using the degenerate peroxidase-specific primer and the vector primer T₇ flanking the cloning site. Four cDNAs, designated GmEpa1, GmEpa2, GmEpb1, and GmEpb2, contained 1298, 1326, 1171, and 1145 nucleotides, excluding poly(A) tail, and encoded mature proteins of 303, 303, 292, and 292 amino acids, respectively. The four predicted amino acid sequences showed homology to other peroxidases. GmEpa1 and GmEpa2 exhibited 97% amino acid identity, GmEpb1 and GmEpb2 exhibited 93% amino acid identity, and GmEpa1 and GmEpb1 exhibited 47% amino acid identity. GmEPa1 and GmEPb1 were expressed as fusion proteins in Escherichia coli. The recombinant fusion proteins were sequestered in inclusion bodies and active forms of the two denatured proteins were recovered after in vitro folding in a medium containing hemin, urea and Ca²⁺. GmEpa1 and GmEpa2 messages were detected in developing seed and root, while GmEpb1 and GmEpb2 messages were present in root, leaf, stem and seed pod. These cDNAs and cDNA-specific primers will allow investigations into peroxidase’s role in development, stress response and in other physiological processes.     

Molecular cloning, expression, and characterization of dnaK in Streptococcus pneumoniae

DnaK is known to be highly conserved in all species and is a major immunogen in Streptococcus pneumoniae. To elucidate the role of dnaK in S. pneumoniae, dnaK was cloned in Escherichia coli using a homologous dnaK probe generated by PCR. The His-tagged DnaK was overexpressed in soluble form and purified from E. coli. Alignment of the deduced DnaK amino acid sequence from nucleotide sequences of the cloned dnaK revealed high homology with DnaK analogs in E. coli (53%) and Staphylococcus aureus (73%). However, anti-pneumococcal DnaK antiserum did not crossreact with DnaK analogs in E. coli, S. aureus and human cells suggesting that pneumococcal DnaK might be a good candidate as a vaccine.     

Molecular cloning and characterization of the alkB gene of Escherichia coli

Summary Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000. Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E. coli chromosome.Abbreviations Ap ampicillin - Cm chloramphenicol - HPLC high performance liquid chromatography - kb kilobases - kd kilodaltons - MMS methylmethane sulfonate - MNU methylnitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - Tc tetracycline - SDS sodium dodecyl sulfate     

Molecular cloning and characterization of the beta-amylase gene from Bacillus circulans

A gene encoding the beta-amylase of Bacillus circulans was isolated from a lambda library and sequenced. The structural gene consists of a 1725 bp open reading frame encoding a polypeptide with a predicted molecular wt of 62830 Daltons. Two active forms of the enzyme were found when the gene was expressed in E. coli. The larger 60 kD form was approximately 3 kD larger than the mature beta-amylase secreted from B. circulans, suggesting that processing of this protein is different between the two species. The smaller 49 kD form is also present at a low level in B. circulans and may result from proteolytic cleavage. The enzyme has a temperature optimum of 50 degrees C. Two other genes, one encoding an alpha-amylase and one a pullulanase, were also isolated from the lambda library.     

Molecular cloning and characterization of the polyphenol oxidase gene from sweetpotato

Polyphenol oxidase is the enzyme responsible for enzymatic browning in sweetpotato that decreases the commercial value of sweetpotato products. Here we reported the cloning and characterization of a new cDNA encoding PPO from sweetpotato, designated as IbPPO (GeneBank accession number: AY822711). The full-length cDNA of IbPPO is 1984 bp with a 1767 bp open reading frame (ORF) encoding a 588 amino acid polypeptide with a calculated molecular weight of 65.7 kDa and theoretical pI of 6.28. The coding sequence of IbPPO was also directly amplified from the genomic DNA of sweetpotato that demonstrated that IbPPO was an intron-free gene. The computational comparative analysis revealed that IbPPO showed homology to other PPOs of plant origin and contained a 50 amino acid plastidial transit peptide at its N-terminal and the two conserved CuA and CuB copper-binding motifs in the catalytic region of IbPPO. A highly conserved serine-rich motif was firstly found in the transit peptides of plant PPO enzymes. Then the homology based structural modeling of IbPPO showed that IbPPO had the typical structure of PPO: the catalytic copper center was accommodated in a central four-helix bundle located in a hydrophobic pocket close to the surface. Finally, the results of the semiquantitative RT-PCR analysis of IbPPO in different tissues demonstrated that IbPPO could express in all the organs of sweetpotato including mature leaves, young leaves, the stems of mature leaves (petioles), the storage roots, and the veins but at different levels. The highest-level expression of IbPPO was found in the veins, followed by storage roots, young leaves and mature leaves; and the lowest-level expression of IbPPO was found in petioles. The present researches will facilitate the development of antibrown sweetpotato by genetic engineering. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1006–1012. The article was submitted by the authors in English.