In this study, about 350 bp cDNA fragment was amplified by PCR. After being sequenced, the AE1-c-end gene fragment was cloned into EcoR I-Pst I site of pGADT7 to form AD ends in the yeast two-hybrid system. The recombinant plasmid was transformed into yeast AH109, and the expression in the yeast was observed. The results demonstrate that AE1-c-end was obtained. pGADT7-AE1-c-end has no toxic effect on the yeast. It can serve as a target gene of yeast two-hybrid system.
Cloning, Molecular ; DNA, Complementary ; Plasmids ; Polymerase Chain Reaction ; Two-Hybrid System Techniques ; Yeasts

Alcohol Oxidoreductases ; genetics ; isolation & purification ; Cloning, Molecular ; Hep G2 Cells ; Humans ; Two-Hybrid System Techniques

No abstract available.
Cloning, Molecular* ; Humans* ; Oxidoreductases*

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

OBJECTIVETo investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).

METHODSYeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.

RESULTSAmong twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.

CONCLUSIONSGenes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins ; genetics ; Cloning, Molecular ; Hepacivirus ; genetics ; Hepatocytes ; metabolism ; Humans ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; Viral Proteins ; genetics

OBJECTIVETo screen and clone the genes of proteins in hepatocytes interacting with hepatitis B virus (HBV) PreS2 by yeast-two hybridization technique.

METHODSThe HBV PreS2 gene was amplified by polymerase chain reaction (PCR) and HBV PreS2 bait plasmid was constructed by using yeast-two hybridization system 3, then transformed into yeast AH109, followed by mating with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) and synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) containing X-alpha-gal for selecting positive blue clones, then amplified by PCR, sequenced, and performed bioinformatics analysis.

RESULTSHBV PreS2 gene was cloned successfully and expressed in yeast AH109.Twenty-six positive colonies were selected, among them, twelve containing metallothionein 2A, one cytochrome C oxidase II, two cytochrome P450 subfamily IV4F, two cytochrome c oxidase subunit 4 isoform 1, three albumin (ALB), one Na(+)K(+) transporting ATPase beta-1 polypeptide, two prealbumin, one lectin galactoside-binding subunit, and Two new genes with unknown function.

CONCLUSIONGenes of HBV PreS2 interacting proteins have been successfully cloned, which brings some new clues for studying the biological functions of HBV PreS2 and related proteins.

Cloning, Molecular ; Hepatitis B Surface Antigens ; genetics ; physiology ; Plasmids ; Protein Precursors ; genetics ; physiology ; Two-Hybrid System Techniques ; Yeasts ; genetics

Cloning, Molecular ; DNA ; genetics ; DNA-Binding Proteins ; genetics ; Humans ; Proteins ; genetics ; Sequence Analysis, DNA ; Two-Hybrid System Techniques ; Yeasts ; genetics

Aims: Expression of recombinant proteins across a range of different host organisms has profound contribution to the advancement in biotechnology. In this study, we aimed to construct a highly versatile broad host range (BHR) expression vector, designated as pYL101C. Methodology and results: The Golden Gate cloning approach was used to construct pYL101C. Key features of pYL101C include a strong integron promoter (PINTc), a BHR pBBR1 origin of replication (ori), gentamycin resistance gene (GmR) as a selectable marker and a multiple cloning site (MCS) downstream of the promoter for easy-cloning purpose. To verify the functionality of pYL101C, we cloned the superfolder green fluorescent protein (sfGFP) reporter gene into pYL101C and transferred the resultant recombinant plasmid pYL101C::sfGFP into various Gram-negative bacteria. Transformants obtained stably expressed strong green fluorescence under blue light excitation even without selection after four passages. Conclusion, significance and impact of study The constructed BHR expression vector, pYL101C and recombinant pYL101C::sfGFP are stable and can be used to monitor the presence of Gram-negative bacteria, such as endophytes and pathogens in their hosts and environment.
Host Specificity ; Plasmids ; Cloning, Molecular

OBJECTIVETo screen and clone hepatocyte protein interacting with hepatitis C virus NS5ATP4A protein for studying its biological functions.

METHODSBait plasmids of hepatitis C virus NS5ATP4A were constructed. After verifying that hepatitis C virus NS5ATP4A protein could be steadily expressed in AH109 yeast strain, yeast-two hybrid assay was performed by mating AH109 with Y187 which pre-transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X-a-gal activity. Nineteen yeast colonies which grew on QDO and had a-gal activity were obtained, and then the library plasmids were extracted and sequenced.

RESULTSSeven genes were screened out and one of them was a formerly unknown gene. They were associated with RNA synthesis, protein translation, cell cycling and tumor immunity.

CONCLUSIONNS5ATP4A binding proteins were successfully screened, which offers new clues for further studying the signal transduction pathway of NS5ATP4A and the pathogenic mechanism of HCV.

Base Sequence ; Cloning, Molecular ; Gene Library ; Genome, Viral ; Hepacivirus ; metabolism ; Hepatocytes ; metabolism ; Humans ; Molecular Sequence Data ; Protein Binding ; Sequence Homology ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; metabolism

To construct the ss/HBsAg protein gene-engineering vaccine for developing the diagnosis and cure tumors in clinical medicine and promoting the growth in animal husbandry production. A pair of primers were designed according separately to the sequence of Somatostatin gene(S14) and HBsAg gene. Their gene fragments were separately amplified by using PCR and cloned, following sequencing, the DNA fragments were inserted into pBluescript vector. Then the ss/HBsAg chimera was constructed and was cloned into pPICZaA plasmid, and transformed into electroporated Pichia pastoris. High yield protein expression was obtained. Expressed protein was proved with high specificity and it's molecular weigh was about 28 KD identified by SDS-PAGE and Western blot.
Artificial Gene Fusion ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Hepatitis B Surface Antigens ; genetics ; Pichia ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Somatostatin ; genetics