Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

Cadherins belong to one of the families of animal glycoproteins responsible for cal-cium-dependent cell-cell adhesion.Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is re-lated to insect’s resistance to Cry1A.The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm,which is 5581 bp in full-length,encoding 1730 amino acid residues(BtR-harm was deposited in GenBank and the accession number is AF519180).Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23,respectively.The inferred amino acid sequence includes a signal sequence,11 cadherin repeats,a membrane-proximal region,a transmem-brane region and a cytoplasmic region.Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2%identity and highly similar to three other lepidopteran cadherin from Bombyx mori,Manduca sexta and Pectinophora gossypiella,with the sequence identities of 60.3.6%,57.5%and 51.0%,respec-tively.The cDNA encoding cadherin gene was expressed successfully in E.coli and the recom-binant proteins can bind with Cry1Ac.Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence,which lo-cated between amino acid 1217 and 1461.Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H.armigera,very low expressed in foregut and hindgut and was not expressed in other tissues.After H.armigera producing resistance to Cry1Ac,the expression quantity of BtR-harm significantly decreased in midgut of H.armigera.It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H.armigera and the binding region was located on a contiguous 244 amino acid sequence,suggesting that the de-crease of expression quantity of BtR-harm is one of the main reasons for H.armigera resistance to Cry1Ac.

Cloning and heterologous expression of pro-2127,a gene encoding cold-active protease from Pseudoalteromonas sp.QI-1

The psychrotropic bacterium, Pseudoalteromonas sp. QI-I, which produces extracellular cold-active protease, was isolated from Antarctic seawater. The genomic DNA of this bacterium was used to construct a plasmid genomic library with the goal of screening cold-active protease genes. Gene pro-2127 with an open reading frame of 2127 bp encoding protease PRO-2127 was cloned and sequenced. Alignment of amino acid sequences suggested that the precursor of PRO-2127 was a member of subfamily S8A, and that it might contain four domains： a signal peptide, an N-terminal prosequence, a catalytic domain and a C-terminal extension. Amino acids Asp185, His244 and Ser425 might form a catalytic triad. PRO-2127 showed some structural features common to psychrophilic enzymes, such as a decrease in Arg residues and the Arg/（Arg＋Lys） ratio. Heterologous expression of pro-2127 in Escherichia coli BL21 （DE3） by pColdlII was also successfully observed in this study.

Cloning and expression of Hsp22.4 gene from Chaetomium globosum

A study was conducted on the molecular mechanism of small heat shock proteins （sHSPs） in Chaetomium globosum. Heat shock protein 22.4 （Hsp22.4） from C. globosum was cloned and expressed in Escherichia coli. BlastX analysis revealed that the Hsp22.4 gene from C. globosum shared the highest identity in amino acid sequence with a Hsp gene from Neurospora crassa, and the identity between them was 65%. The C. globosum Hsp22.4 gene was inserted into the expressive vector of pGEX-4T-2 and the recombinant plasmid named pGEX-HSE E. coli BL21 transformed with pGEX-HSP plasmid was induced by IPTG, and the expressed proteins were analyzed with SDS-PAGE. A 50 kD protein was specially expressed in E. coli BL21, and the result was consistent with expectation, and showed that the Hsp22.4 gene had been expressed in E. coli. Our study has made a foundation for further studying the function ofsHSPs protein.

Construction of recombinant plasmid and prokaryotic expression in E. Coli and biological activity analysis of human placenta arresten gene

BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerpera's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5α, BL21 and BL21 (DE3) by the CaCl_2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purlfled, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E. coil have laid a foundation for further study of its anti-angiogenic function and may pave the way for future antitumor application.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

Cloning of Chinese obese cDNA and its expression in E coli

Objective To obtain the sequence of Chinese obese (OB) cDNA and establish a method of leptin production in China Methods Han Chinese OB cDNA fragment was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and was inserted into the expressing vector pBV220 Then the constructed recombinant plasmid pBV220 OB was transformed to E coli DH5α for leptin expression The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression E coli cells were lysed by high pressure homogenization After cell membrane was extracted, the inclusion bodies were mainly renatured and purified primarily by precipitation with ammonium sulfate and gel chromatography through a Sephadex G75 column The activity of recombinant leptin was determined by its influence on the satiety and weight gain of mice Results Analysis of DNA sequence showed that Han Chinese OB cDNA included the glutamine codon at 49 The amount of recombinant leptin expressed in E coli accounted for 31%-47% of total cellular proteins From 1?L of fermentative bacteria about 40?mg of pure recombinant human leptin was isolated with a purity of being above 95% The recombinant human leptin could reduce food intake and inhibit weight gains in mice Conclusion The glutamine codon at 49 is not missing in Chinese OB gene The biologically active human leptin can be obtained by a relatively simple method of recombinant DNA technology