Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Hepatitis B is one of the most serious global threats to human health. Phylogenetic analysis of hepatitis B virus (HBV) can reveal the evolutionary relationship between HBV sequences and thus provide a basis for the prediction and treatment of hepatitis B and other aspects. In this study, we performed sequence analyses on the HBV sequences of five clinical HBV samples and the HBV sequences retrieved from the GenBank, EMBL, and DDBJ to construct a phylogenetic tree and analyze sequence structures. The experimental results revealed that the C gene of one cloned sequence had a recombinant structure of HBV B/ C subtype. Moreover, the phylogenetic results proved the existence of a newly found subtype HBV/B6 in Xishuangbanna of Yunnan Province, China. The experimental conclusion represents certain value for phylogenetic studies of HBV in Yunnan ethnic minority groups.
DNA, Recombinant ; genetics ; Genes, Viral ; genetics ; Genotyping Techniques ; Hepatitis B virus ; classification ; genetics ; Humans ; Phylogeny

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.
DNA Topoisomerases, Type I ; biosynthesis ; genetics ; Fermentation ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVEThis study was conducted to investigate the function of the first intron of porcine growth hormone (pGH) gene in the gene expression.

METHODSPCR method was used to amplify the first intron from pig genomic DNA. The intron was then inserted into pGH cDNA to construct pGH cDNA-intron (pGH cDNA-in). The recombinant adenoviruses containing pGH cDNA and pGH cDNA-in genes under control of CMV promoter were generated by homologous recombination method in HEK 293 cells respectively. The effect of the first intron on gene expression was evaluated by comparing the expression levels of pGH cDNA-in and pGH cDNA mediated by adenovirus vectors in CHO cells.

RESULTSThe expression level of pGH cDNA containing the first intron increased by 117%, which was significantly higher than that of pGH cDNA without the intron (P < 0.001).

CONCLUSIONThe first intron of pGH gene has the function to improve pGH gene expression.

Adenoviridae ; genetics ; Animals ; CHO Cells ; Cricetinae ; DNA Transposable Elements ; DNA, Complementary ; DNA, Recombinant ; Genetic Vectors ; Growth Hormone ; genetics ; Introns ; Swine

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Plasmids ; genetics ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo construct the recombinant adenovirus vector expressing human endostatin.

METHODSHuman endostatin gene extracted from pGEM-T Easy vector containing the target gene fragment was successfully amplified using PCR and cloned into pShuttle2 vector. The target gene was subcloned into an adenovirus vector and the resulted recombinant adenovirus (Ad-hEndo) was linearized before transfected into HEK 293 packaging cells. The Ad-hEndo recombinant adenovirus was efficiently amplified in 293T cells and purified by CsCl density centrifugation, and the titer of the virus was determined.

RESULTSThe amplified hEndostatin cDNA was verified by PCR and sequencing, and the resulted virus titer reached 5.2 x 10(9) pfu/ml.

CONCLUSIONThe recombinant adenovirus containing human endostatin gene has been successfully constructed, which may provide important basis for gene therapy research for angiogenesis-dependent diseases.

Adenoviridae ; genetics ; Cell Line ; Cloning, Molecular ; DNA, Recombinant ; genetics ; Endostatins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

BACKGROUNDTo express in vitro the bovine enterokinase catalytic subunit (EKL) protein, which could be used in the future for the cleavage and purification of fusion proteins.

METHODSBovine enterokinase catalytic subunit cDNA was obtained by RT-PCR from duodenal mucosa of a bovine obtained at wholesale market, and then cloned into a pUCmT cloning vector and sequenced. The desired gene fragment was inserted into a pET39b expression plasmid and the recombinant vector pET39b-EKL was transformed into E. coli BL21 (DE3). Protein expression was induced using IPTG. The recombinant DsbA-EKL was purified with His.Tag affinity chromatography, and it bioactivity was analyzed.

RESULTSCompared with the sequence deposited in GenBank, the sequence of the EKL gene cloned in the present study is correct. It was also confirmed that the nucleotide sequence of expression plasmid pET39b-EKL was correct at the conjunction site between the recombinant DNA 5' terminal multi-cloning site and the recombinant fragment. SDS-PAGE analysis indicated that the target product was about 65 kDa and represented 28% of total cell protein. Purified recombinant protein was obtained by metal chelating chromatography using Ni-IDA resin. After desalting and changing the buffer, the crude kinase was incubated at 21 degrees C overnight and shown to have a high autocatalytic cleavage activity.

CONCLUSIONSThe EKL gene from Chinese bovine has been cloned successfully and expressed. This investigation has layed the foundation for future enterokinase activity research and for further large-scale application of expression products.

Animals ; Catalytic Domain ; genetics ; Cattle ; Cloning, Organism ; DNA, Complementary ; Enteropeptidase ; analysis ; genetics ; Recombinant Proteins

OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication

In 2010, the artificial synthesis of Mycoplasma mycoides triggers the new era of synthetic biology. This great breakthrough is achieved mainly thanks to the powerful DNA recombinant ability of yeast. In recent years, except for the methods used for large DNA assembly on the basis of in vivo homologous recombination, various different DNA assembly methods in vitro, based on the concept of DNA ligation or polymerization, have also been developed, such as Biobrick\BglBrick, SLIC and Gibson one-step assembly. Application of these new technologies has greatly accelerated the construction of synthetic part libraries, biosynthetic pathway and even microbial chromosomes. In fact, all DNA assembly methods are derived from the combinations of DNA joining and organizational schemes. This review describes the brief introduction of the main in vivo and in vitro DNA assembly protocols developed so for, which will benefit the construction of different types of synthetic functional devices and also biosynthetic pathways in the research of synthetic biology in China.
DNA ; biosynthesis ; chemistry ; genetics ; DNA, Recombinant ; biosynthesis ; genetics ; Genetic Engineering ; methods ; Metabolic Networks and Pathways ; Synthetic Biology ; methods

Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02), and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the alpha-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut+ carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction, the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7 u/mg.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Superoxide Dismutase ; biosynthesis ; genetics