The cDNA of cattle Ghrelin gene was amplified from abomasum fundic gland mRNA of Qinchuan Cattle by RT-PCR. PCR product was cloned into the T vector pGM-T to construct pGh-T1 for sequencing. Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET32a (+) and transformed into host Escherichia coli strain BL21 (DE3) for expression. The expression of pGh-32 mature Ghrelin protein was induced by IPTG and was identified by SDS-PAGE. The expression product was observed with soluble protein and inclusion body. Western blotting showed that the recombinant protein was recognized by his-antibody specifically. The protein was purified by Ni-NTA column and was used to inject rabbits to obtain polyclona antibody. ELISA result showed that the antibody titer was 1:12 800. The immunohistochemistry test between the hypothalamus arcuate nucleus and the antibody showed that fusion protein had biological activity. This will provide a basis for further study on the biological function of Ghrelin protein to growth and development and fat deposition of cattle.
Animals ; Cattle ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Ghrelin ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

The fused gene (PTH-TFN) of parathyroid hormone (PTH) gene and transferring N-terminal half-molecule (TFN) gene was amplified by multiple PCR and inserted into pPIC9 vector. The recombinant plasmid pPIC9-PTH-TFN was transformed into Pichia pastoris GS115 by PEG. After methanol induction, the target protein was expressed in fermentation supernatant at high level. The fused protein PTH-TFN with purity being higher than 95% was finally obtained after purification through two-step chromatography: SP Sepharose Fast Flow and Phenyl Sepharose Fast Flow. Western blot analysis and adenylate cyclase assay proved that the fused protein exhibited the bioactivity to stimulate cAMP synthesis and the ability to bind Fe3+ in the Fe3+ saturation study as the recombinant TFN did indicating that TFN could be used as the transcellar carrier of PTH.
Artificial Gene Fusion ; Cloning, Molecular ; Humans ; Parathyroid Hormone ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transferrin ; genetics

Ubiquitin-ribosomal protein S27a(UBRPS27a) is a fusion protein of Ubiquitin and ribosomal protein. The N-terminal is ubiquitin and C-termina is ribosomal protein S27a with a high conservative zinc finger domain of the C2-C2 type. When it was expressed in eukaryotes,The intact fusion protein were rapidly processed to free ubiquitin monomer and ribosomal protein S27a (RPS27a). Ubiquitin degradated proteins particularly and selectively in cell and RPS27a is indispensable for translation. This multifunctional ribosomal protein is expressed at high levels in a wide variety of actively proliferating cells and tumor tissues and is a representative characteristic of various tumor cells. In our preliminary study of this protein in the silkworm,RPS27a also be found express highly in actively proliferating cells. The precise functional role of each ribosomal protein is largely unknown and many ribosomal proteins have extraribosomal functions apart from the particle. In this article, we review the recent research on the connection between tumor and this fusion protein, Ubiquitin-Proteasome Pathway and ribosomal protein. These research may indicate the origin and development of tumor, provide the basis for clinical diagnosis of cancer and the novel therapeutic targets for the treatment of malignant tumors.
Animals ; Biomarkers, Tumor ; Cloning, Molecular ; Humans ; Neoplasms ; genetics ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Ribosomal Proteins ; biosynthesis ; genetics ; Ubiquitin ; biosynthesis ; genetics

OBJECTIVETo enhance the expression level of human papillomavirus (HPV) 16 L2E7 in Escherichia coli (E. coli), in aim of providing high-level expression of HPV16 L2E7 strain for pre-clinical high-throughout production.

METHODSThe whole L2E7 gene was optimized by software of Synthetic Gene Designer, reflecting E. coli codon usage. Two parts of codon-optimized gene were cloned into pET9a vector step by step. The positive clone, which was sequenced to be corrected, was transfected to BL21 (DE3+) via isopropyl-beta-D-thiogalactoside (IPTG) induction. They produced the HPV16 L2E7 fusion protein, which was further detected by SDS-PAGE and Western blot. The induction temperature, induction time, and IPTG concentration were also optimized by a series of experiments. Further purification modes of this protein were also explored.

RESULTSCodon-optimized HPV16 L2E7 was highly expressed in E. coli. The target protein accounted for nearly 60% of the total cell extract.

CONCLUSIONHigh-level expression of HPV16 L2E7 was successfully constructed.

Codon ; Escherichia coli ; genetics ; metabolism ; Human papillomavirus 16 ; metabolism ; Papillomavirus E7 Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Foot-and-mouth disease virus (FMDV) is the aetiological angent of a highly contagious viral disease. The complete gene encoding the structural protein of FMDV (P1) was subcloned into expression vector pGEX-KG, resulting in the fusion expression plasmid pKG-P1. After transformed into E. coli BL21(DE3) and induced by IPTG, the results of SDS-PAGE showed that the GST-P1 fusion protein was expressed in high level. The molecular weight of the fusion protein wa 110kD and the expressed products were soluble. Western-blotting was performed to confirm that the expressed fusion protein could specifically react with antiserum against FMDV. The fusion proteins were further purified by GST purification kit and an indirect ELISA (P1-ELISA) based on the purified proteins was developed. Comparison between P1-ELISA and the standard indirect haemagglutinin assay showed the two methods had 87 per cent agreement by detecting 864 serum samples, indicating the purified P1 protein was specific as the antigen of indirect P1-ELISA.
Capsid Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease Virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

To understand the function of Mayven and investigate the pathogenesis of multiple sclerosis, the gene sequences of different truncated Mayven were amplified from the gene library of human brain. These truncated fragments, including fragment P1 (1-902 bp), fragment P2 (1-523 bp), fragment P3 (507-182 bp) and fragment P4 (887-1782 bp), were cloned into pGEX-4T-2 vector to construct recombinant plasmids. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The expressed proteins were detected by SDS-PAGE and Western blot, and were purified by GST purifying system. The results showed that recombinant express vectors of different truncated GST-Mayven were successfully constructed and were expressed in soluble form protein induced by IPTG. The fusion proteins have good reactivity to GST antibody. The construction of recombinant express vectors of different truncated GST-Mayven lays a basis for further function study on Mayven.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Multiple Sclerosis ; genetics ; Nerve Tissue Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

Human Sodium/Iodide symporter gene cDNA was amplified from thyroid tissue of the patient suffering from Graves disease by RT-PCR, and T/A cloned into pGEM-TEasy-NIS for sequencing, subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene, and then forwarded to homologous recombinant in the bacteria BJ5183 that already contained AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the 293 cells to obtain recombinant adenovirus. The results showed that the recombinant AdNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 2. 5 - 3 x 10(9) efu/ml. So, the recombinant adenovirus vector carrying hNIS was successfully constructed, thus providing a basis for researches on 131I therapy in nonthyroid carcinoma.
Adenoviridae ; genetics ; metabolism ; DNA, Complementary ; genetics ; Genetic Vectors ; genetics ; metabolism ; Graves Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Symporters ; biosynthesis ; genetics

Wheat grain peroxidase 1 (WP1) belonged to class III plant peroxidase with cofactor heme, which not only has antifungal activity, but also influences the processing quality of flour. In order to enhance functional expression of WP1 in prokaryotic system by increasing endogenous heme synthesis, we constructed a recombinant plasmid pACYC-A-L containing hemA and hemL of Esherichia coli. Then, we co-transformed it into host strain T7 Express with secretive expression vector (pMAL-p4x-WP1) or non-secretive expression vector (pET21a-MBP-WP1), respectively. The MBP-WP1 fusion protein was further purified by amylose affinity chromatography and its peroxidase activity was assayed using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate. At 12 h after induction at 28 degree, the extracellular 5-aminolevulinic acid (5-ALA) production of T7 Express/pACYC-A-L was up to 146.73 mg/L, simultaneously the extracellular porphrins also increased dramatically. The peroxidase activity of functional MBP-WP1 obtained from T7 Express/ (pACYC-A-L + pMAL-p4x-WP1) was 14.6-folds of that purified from T7 Express/ pET21a-MBP-WP1. This study not only successfully enhanced functional expression of wheat peroxidase 1 in Esherichia coli, but also provided beneficial references for other important proteins with cofactor heme.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Heme ; biosynthesis ; genetics ; Peroxidases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transformation, Genetic

BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.

METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.

RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.

CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo express SPAG4L, a novel human testis gene in E. coli and purify it's fusion protein.

METHODSThe fragment encoding SPAG4L126-379 was amplified by RT-PCR and the PCR products were cloned into PUCm-T vectors. After digestion by EcoR I and Hind III, the fragment was subcloned into PQE-30, a prokaryotic expression vector with 6×His tag. The recombinant plasmid PQE-30-SPAG4L was sequenced and transformed into E.coli M15. The expression of his-tagged fusion protein was induced by IPTG. The fusion protein was identified by Western blotting and purified using Ni-NTA magnetic agarose beads.

RESULTSThe recombinant plasmid PQE-30-SPAG4L was constructed successfully and expressed in E.coli M15. The fusion protein SPAG4Lwith 6×his-tag was confirmed by Western blotting. The micro-scale purification system of 6×His-tagged SPAG4Lprotein was established and purified fusion protein was obtained.

CONCLUSIONThe recombinant plasmid PQE-30-SPAG4L can be expressed in vitro and used for studying the biological function of SPAG4L in spermatogenesis.

Carrier Proteins ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Humans ; Male ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification