Two transgenic mouse strains, in which the expression of human factor IX (hFIX) in the milk were different significantly, were bred, and the foreign gene integration as well as the content of hFIX in the milk were detected by PCR, Southern blot, FISH and ELISA, respectively. The results showed that approximately 50% offsprings were transgenic positive. Foreign gene integrated in mouse chromosomes was intact. The hFIX expression of each mouse in the same strain was different, the content of hFIX in the milk was (43.32 +/- 5.41) microgram/mL in FIX-33 transgenic strain and (1.16 +/- 0.45) microgram/mL in FIX-124 transgenic strain. Meanwhile, the hFIX gene expression between the two strains was different remarkably (P < 0.01). We conclude that the characteristics of inheritance and expression in the founder were able to be transferred to their offsprings stably.
Animals ; Factor IX ; analysis ; biosynthesis ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Mice ; Mice, Transgenic ; Milk ; chemistry ; Recombinant Proteins ; biosynthesis

OBJECTIVETo perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli.

METHODSSjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition.

RESULTSThe RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM).

CONCLUSIONThe engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; analysis ; chemistry ; Escherichia coli ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; Schistosoma japonicum ; genetics ; Tropomyosin ; biosynthesis ; chemistry ; genetics

We used reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) techniques to obtain the full-length cDNA of beta-mannanase (EC 3.2.1.78) from Armillariella tabescens EJLY2098 (an edible fungus). Sequence analysis of the 1481 bp full-length cDNA encoding 445 amino acid residues indicated that the gene contained two structural domains, cellulose-binding domains (CBD) and glycoside hydrolase family 5 (GHF5) domains, other than the conserved beta-mannanase domain. Thus, we classified this gene as a member of glycoside hydrolase family 5. Next, we cloned a 1308 bp fragment encoding the beta-mannanase mature peptide (re-atMAN47) into the expression vector pPICZalphaA and expressed it in Pichia pastoris. The yield was 440 mg/L. Enzyme activity reached a maximum of 1.067 IU/mL after 72 h of methanol induction. The re-atMAN47 had an optimal temperature of 60 degrees C and an optimal pH of 5.5. It manifested broad thermostability from 30 degrees C-65 degrees C, and was stable between pH 4.5-7.0. This study represents the first record of a beta-mannanase from Armillariella tabescens EJLY2098 and provides a new source of carbohydrate hydrolysis enzyme with good biosafety, thermostability and wide pH stability. It is a good approach for the industrial needs of feed, food and pharmaceutical manufacturers.
Armillaria ; classification ; enzymology ; genetics ; Cloning, Molecular ; Enzyme Stability ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; beta-Mannosidase ; biosynthesis ; chemistry ; genetics

Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbate-glutathione recycling reaction. In this study, two DHARs genes (SmDHAR1 and SmDHAR2) were isolated from Selaginella moellendorffii. The SmDHAR1 and SmDHAR2 genes encode two proteins of 218 and 241 amino acid residues, with a calculated molecular mass of 23.97 kDa and 27.33 kDa, respectively. The genomic sequence analysis showed SmDHAR1 and SmDHAR2 contained five and six introns, respectively. Reverse transcription PCR revealed that the SmDHAR1 and SmDHAR2 were constitutive expression genes in S. moellendorffii. The recombinant SmDHAR1 and SmDHAR2 proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The recombinant SmDHAR1 showed 116-fold higher enzymatic activity towards the substrate dehydroascorbate than recombinant SmDHAR2. The recombinant SmDHAR1 showed higher thermal stability than recombinant SmDHAR2. These results indicated obvious functional divergence between the duplicate genes SmDHAR1 and SmDHAR2.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Plant Proteins ; biosynthesis ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Selaginellaceae ; enzymology ; genetics ; Sequence Analysis, DNA

To study the mechanism of thrombogenesis and search new anti-thrombotic agent, the cDNA for human vWF A1 domain was high-level expressed in E. coli and recombinant protein of vWF A1 with biologic activity was obtained. The gene encoding A1 domain was amplified by PCR from plasmid containing full length cDNA of human vWF. After confirming by DNA sequencing analysis, the recombinant expression plasmid pQE31-vWF/A1 was constructed and introduced into E. coli M15 strain, then induced by IPTG; the expressed protein was purified with Ni-NTA agarose, identified by Western blotting. The results showed that the 854 bp DNA fragment was obtained by PCR from the plasmid containing full length cDNA for human vWF and its sequence was identical to the published sequence. High level expression of A1 protein was yielded after 5 hour-induction, which amounted to 30% of total bacteria protein in inclusion body. Western blot demonstrated it possessed good antigenicity and high specificity. It is concluded that cDNA for vWF/A1 had been cloned successfully, high level expression of A1 protein was achieved in E. oli. This study will provide a basis for the further clinical and basic research on the role of vWF in thrombosis and hemostasis.
Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Escherichia coli ; genetics ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; analysis ; von Willebrand Factor ; analysis ; biosynthesis ; genetics

This study was aimed to construct the soluble HLA-A*0201-PR1 complex for preparation of HLA-A*0201-PR1 tetramer. The recombinant HLA-A*0201-BSP (BirA substrate peptide) fusion protein as heavy chain and beta(2)-microglobulin (beta(2) m) as light chain were expressed highly as insoluble aggregates in Escherichia coli and then purified with gel filtration, and the final purity reached above 90%. The two subunits were refolded to form an HLA-A*0201-peptide complex by dilution method in the presence of an antigenic peptide PR1, a HLA-A2-restricted peptide from proteinase 3 (aa 169 - 177, VLQELNVTV). Refolded HLA-A*0201-PR1 complex was biotinylated using a BirA enzyme and purified by anion exchange chromatography on a Q-Sepharose (fast flow) column. The extent of reconstitution of the HLA-A*0201-PR1 complex was analyzed by HPLC gel filtration. The refolded and biotinylated products were detected by Western blot and ELISA with monoclonal antibody BB7.2 that recognized the natural conformations of HLA-A2 and streptavidin. The results showed that the refolded complex was composed of HLA-A*0201-BSP aggregate, HLA-A*0201-PR1 complex and beta(2) m, and reconstitution yields of 18% with PR1 was obtained. Refolded HLA-A*0201-PR1 complex could be confirmed by practical immunological method and biotinylated efficiently. It is concluded that the refolding and biotinylation of HLA-A*0201-PR1 complex is successfully obtained. This work provides the basis for the preparation of HLA-A*0201-PR1 tetramer.
DNA Primers ; genetics ; Escherichia coli ; genetics ; HLA-A Antigens ; analysis ; biosynthesis ; genetics ; HLA-A2 Antigen ; Humans ; Oligopeptides ; genetics ; metabolism ; Protein Binding ; Protein Folding ; Recombinant Fusion Proteins ; analysis ; biosynthesis ; beta 2-Microglobulin ; biosynthesis ; chemistry ; genetics

This experimental study was aimed to construct the recombinant bisbicistronic eukaryotic expression vector containing endocrine and exocrine protein (EECP) gene associated with breast cancer and enhanced green fluorescent protein (EGFP) gene. And then we transfected it into breast cancer cells MCF-7 to detect the expression of EECP protein and study preliminary biological function of EECP gene. The EECP sequence was cloned to pBluescript II SK (+) plasmid. After restriction endonuclease reaction of pBluescript II SK(+) plasmid, the EECP fragment was cloned to pIRES2-EGFP vector forming a recombinant eukaryotic expression vector named pEECP-IRES2-EGFP. The potential vector was identified by restriction endonuclease digestion and sequencing. Correct plasmid was extracted and transfected into breast cancer cells MCF-7. The expression of EECP protein was detected by western blot analysis. Its biological function was studied by MTT and Flow-cytometry. It turns out that the recombinant eukaryotic expression vector containing EECP gene and EGFP gene was constructed successfully, and it could transfect MCF-7 cells efficiently. It can get higher expression of EECP protein and higher cell proliferation, thus providing an important and convenient tool for studying the function of EECP gene in vitro and in vivo.
Base Sequence ; Breast Neoplasms ; genetics ; pathology ; Female ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; MCF-7 Cells ; Molecular Sequence Data ; Proteins ; analysis ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Ribosomes ; chemistry ; metabolism

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

The CBD gene from Trichoderma reesei was cloned into the Corynebacterium glutamicum secretion expression vector pXMJ19-sp, in which green fluorescent protein was inserted to obtain pXMJ19-sp-GFP-CBD. After induced by 0.5 mmol/L IPTG, GFP-CBD was expressed in Corynebacterium glutamicum at high level of 200 mg/L. The GFP-CBD could be purified to high purity with cellulose column. The results indicated CBD can be successfully used in Corynebacterium glutamicum expression system and thus offer an extremely simple, effective and scalable way for production of recombinant proteins.
Base Sequence ; Cellulases ; biosynthesis ; genetics ; Cellulose ; chemistry ; genetics ; Cloning, Molecular ; Corynebacterium glutamicum ; genetics ; metabolism ; Cost-Benefit Analysis ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Protein Engineering ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Trichoderma ; genetics

Acetylcholinesterase (AChE) plays a key role in the pesticide determination. However, the extraction of AChE from natural materials has the disadvantages of low yield, complex purification and poor stability. Therefore, the preparation of recombinant AChE with high performance becomes the hot topic of researchers in recent years. In this article we summarize the progress in the expression of recombinant AChE and the improvement of its analytical characteristic. Finally, we point out that the directed evolution strategy combined with surface display technology is the future trend on improving recombinant AChE activity.
Acetylcholinesterase ; biosynthesis ; chemistry ; genetics ; Baculoviridae ; genetics ; metabolism ; Cell Surface Display Techniques ; Cholinesterase Inhibitors ; analysis ; Evolution, Molecular ; Genetic Vectors ; genetics ; Pesticide Residues ; analysis ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics