ABSTRACT
Objective To construct the prokaryotic expression vector pET30a( +)-VH-mms13 and identification of its protein after induced with IPTG.Method Heavy chain variable region VH gene of typeⅣcollagenase monoclonal antibody and magnetosome membrane protein gene mms13 were amplified separately,the fusion gene VH-linker-mms13 were synthesized by SOE-PCR technique and inserted into pET30a ( +) plasmid, which was confirmed by restriction enzyme digest and sequencing.Then the recombinant plasmid pET30a ( +)-VH-mms13 was transform into E.coli DE3 and induced with 0.4 mmol/L IPTG.The fused protein was identified by SDS-PAGE and Western blot.Results The length of fusion gene VH-mms13 was 738 bp,and the sequence was correct.After induced with IPTG,the fused protein was found in the inclusion body and Western blot results suggested that the fused protein can bind with His-tag antibody specifically.Conclusion Expression vector pET30a ( +)-VH-mms13 is successfully constructed and the fusion protein has good immunogenicity,which lay the foundation for the development of biomagnetism-targeted drug.
ABSTRACT
Phytic acid, the major storage form of phosphorus in plant seeds is degraded by the phytases to yield inositol and free phosphate, contributing thereby to the improved bioavailability of phytate phosphorus and essential minerals in plant foods and simultaneous reduction in phosphorus pollution of the terrestrial and aquatic ecosystems. As a possible strategy for altering seed phytate levels, the approach involving reduction of phytate content by ectopically expressing endogenous phytase gene during seed development of soybean (Glycine max L. cv. Pusa-20) was attempted in the present study. Semi-quantitative RT-PCR revealed the maximum expression of phytase gene transcripts in germinating cotyledons (~10 days after germinations), compared to other vegetative tissues. A full-length phytase cDNA was amplified from the germinating seedlings by splicing by overlap extension (SOE)-PCR and its sequence analysis revealed an open-reading-frame of 1644 bp, including an N terminal signal peptide of 28 amino acids. Predicted amino acid sequence (547-aa) of molecular mass 62 kDa on alignment with related purple acid phosphatases in other plants shared five conserved domains and seven invariant amino acids involved in coordination of the metals in the binuclear center of purple acid phosphatases. Owing to a large number of E. coli low-usage codons in soybean phytase gene, the modified gene was cloned into a prokaryotic expression vector pET-28a (+) and its expression in E. coli was confirmed by SDS-PAGE and Western blot analysis. Bioassay of the crude expression product in E. coli revealed a functional phytase gene, showing a great potential for developing low phytate transgenic soybean through its seed-specific overexpression in the early stages of seed development.
Subject(s)
6-Phytase/biosynthesis , 6-Phytase/chemistry , 6-Phytase/genetics , Amino Acid Sequence , Cloning, Molecular , Codon/genetics , DNA, Complementary/genetics , Escherichia coli/genetics , Gene Expression , Gene Expression Regulation, Plant , Genetic Engineering/methods , Minerals/metabolism , Molecular Sequence Data , Organ Specificity , Phosphorus/metabolism , Phylogeny , Seedlings/genetics , Sequence Homology , Glycine max/enzymology , Glycine max/genetics , Glycine max/metabolismABSTRACT
The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.
Subject(s)
Humans , Animals , Erythropoietin , Glycosylation , Pichia/genetics , Polymerase Chain ReactionABSTRACT
Objective To construct and express a small complement receptor type 1 (CR1) deriv-ative which contained two functional domains. Methods Total RNA was isolated from the peripheral blood mononuclear cell. The functional fragment Ⅰ of CR1 was amplified using the RT-PCR. The functional frag-ment Ⅱ was amplified with the plasmid of pET-32a-CR1-SCR15-18 as template which had been already con-structed in our laboratory. Then the chimeric gene that contained the two fragments was constructed with spli-cing overlap extention PCR. The chimeric gene was then inserted into the plasmid of pET-32a (+) and transformed into E. coli Rosetta(DE3). The inserted gene was verified by enzyme digestion and DNA se-quencing. After induced by IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot. Then the fusion protein was purified by Ni-NTA affinity column and renatured through dialysis. The comple-ment inhibition activity was determined by CH50 method. Results The chimeric gene was successfully cloned into pET-32a(+). The result of SDS-PAGE and Western blot confirmed the expressed protein and showed that the molecule mass(Mr) of the expressed protein was 63×103. The purity of the recombinant protein was up to 92% after Ni-NTA column affinity chromatography. The bioactivity assay showed the fusion protein had a concentration-dependent complement inhibition activity within the concentration range of 0-200 μg/ml. Conclusion The two functional domains contained small CR1 derivative was successfully construc-ted and expressed in E. coli Rosetta. The fusion protein had a relative high bioactivity, providing a basis for further function experiment in vivo.
ABSTRACT
Objective To construct an effective expression system for secretory canstatin, and to study its biological effects. Methods Canstatin cDNA was ligated with the cDNA of CEA signal peptide by SOE PCR, and the new fused fragment was cloned into eukaryotic expression vector pCMV-Script. The recombination vector pCMV-CEAS-Cans was transferred into human umbilical vein endothelial cells (HUV-EC) and human lung adeno-carcinoma cell line A549 by cationic liposome. The expression of canstatin mRNA together with CEAS mRNA in the transformed cells were detected by real time PCR method. Results Canstatin mRNA was detected in both transformed cells. The 3 H-TdR intake rate in pCMV-CEAS-Cans transformed HUVEC cells is significant lower than that of the pCMV-Script transformed cells (P