ObjectiveTo study Decorin biological effects,Decorin gene cDNA was cloned and expressed in E. coli. MethodsThe Decorin gene obtained by PCR from T cell cDNA library and cloned into plasmid vector pUC19. The encoding sequence of Decorin in the pUC19 was confirmed by DNA sequencing using Sanger Dideoxy method, and then it was subcloned into expression plasmid vector pGEX-4T-1, the recombinant vector was indentified with BamHI and EcoRI in 1.2 ％ agarose gel electrophoresis. The expression of Decorin fusion protein with GST in E. coli JM109 was induced with IPTG and identified by SDS-PAGE. ResultsThe PCR amplified DNA fragment shared identical sequence with known Decorin gene reported in GenBank (Accession number: AF138300). The SDS-PAGE analysis revealed that the expressed fusion protein MW was approximatelly 65.6KD and in soluble format. ConclusionThe cloned Decorin gene is correct and it was expressed in fusion protein with GST.

The relationship between antiproliferative effect of human IFN-γ-EGF3 fusion protein and the influence of EGF receptor binding activity has been studied on A431 cell line. Antiproliferative activity of human IFN-γ-EGF3 was higher than that of its parent IFN-γ. In the 125 I-EGF receptor competition experiment, the inhibition of EGF receptor binding capacity on the target cells was observed in the treatments of human IFN-γ or IFN-γ-EGF3, but the later was more significant. Our data suggests that the antiproliferative effects by IFN-γ and its fusion protein are closely related to their EGF receptor competitions.

Aim To express and purify a new sub-type of interferon α , α 1c in E.coli. Methods To establish a brief protocol for expression and purification of IFN-α 1c. In vitro assay for the IFN activity is required for the measurement of antiviral, antiproliferative and immunoregulatory properties. Results Expression of IFN-α 1c was verified by its reactivity to IFN-α 1-specific neutralizing antibodies by ELISA. Western blot also detected a band with relative molecular mass(Mr) of 19 000. IFN-α 1c possessed antiviral biological activity(3.2× 107) higher than IFN-α 1b(1.0× 107～ 1.8× 107) did on VSV challenged WISH cells. IFN-α 1c also inhibited growth rate of A549 comparable to IFN-α 1b. IFN-α 1c as an immunologic effector also augmented the cytotoxic activity of NK cells. Conclusion These results demonstrate that IFN-α 1c can be successfully expressed in E.coli. This interferon with high bioactivity may be a good candidate for clinical use.

OBJECTIVETo increase prokaryotic expression level of IFN-alpha 1C gene through the quantitative theory of translational control and the translational enhancer sequence.

METHODSStepwise polymerase chain reaction (PCR) was used to alter the 5 terminal cDNA sequence of IFN-alpha 1C in three different grades of base mutation. In this way, the free energy (Delta G) of the secondary structure in translational initiation region (TIR) was decreased gradually. An expression plasmid (pBVE) was constructed to contain the translational enhancer cDNA sequence by modifying pBV220 upstream of the SD region.

RESULTSThe expression levels of three kinds of IFN-alpha 1C modified gene were all increased. Furthermore, it presented an increasing trend with decreasing in delta G varying from -50,241.6 to -22,190.0 J/mol. The highest expression was 2.43 x 10(8) U/L, covering twelve times more than its original cDNA. IFN-alpha 1C gene and its modified cDNA was inserted into pBVE as reporting genes. E.Coli cells harbouring pBVE/IFN-alpha 1Cs cDNA produced two to five times more IFN than cells harbouring pBV220/IFN-alpha 1Cs.

CONCLUSIONSpBVE containing translational enhancer is a high level prokaryotic expression vector. The theory of quantitative translational control can effectively be used to enhance the IFN-alpha 1C gene expression level in E.coli.

DNA, Circular ; genetics ; Enhancer Elements, Genetic ; Escherichia coli ; genetics ; Gene Expression ; Gene Expression Regulation ; Genetic Vectors ; Interferon Type I ; genetics ; Peptide Chain Initiation, Translational ; genetics ; Polymerase Chain Reaction ; methods

BACKGROUND: Human keratinocyte growth factor-2 (hKGF-2) has extensive physiological functions, which plays an important role in embryonic development, tissue-repairing, nervous regeneration, vascularization and development of tumor.OBJECTIVE: To clone hKGF-2 gene, obtain the expression of hKGF-2 in Escherichia coli(E.coli) and determine its bioactivity, so as to provide experimental basis for further investigation.DESIGN: Open experiment.SETTING: Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.MATERIALS: The experiment was conducted in State Key Laboratory of Viral Genetic Engineering, Institute for Viral Disease Control and Prevention of Chinese Center for Disease Control and Prevention. The temperature control expression vector pBV220 was constructed by State Key Laboratory of Viral Genetic Engineering; EcoR Ⅰ , BamH Ⅰ , T4 DNA ligase (Promega Co., Ltd.); The specific polymerase chain reaction (PCR) of hKGF-2 (Manufactured by Shanghai Boya Biotechnology Co., Ltd.); Heparin-Sepharose CL-6B (Pharmacia Company); PCR rapid purification kit,Trizol kits for total RNA extract, Kits for RT-PCR (GIBCO Co., Ltd.); Kits for rapid extraction of plasmid DNA (Boda Company); BL-21-codon plus compent cells (Stratagene Co., Ltd.).METHODS: High-expression strain BL-21 codon plus competent cells was used to express and purify initially recombinant hKGF-2 protein, and its activity was detected. RT-PCR was adopted to obtain hKGF-2 cDNA from lung tissues of naturally aborted fetus and clone it into pBV220 carri er plasmid. The hKGF-2 protein expressed in BL-21 codon plus competent cells of E.coli. Affinity chromatography and ion exchange chromatography were applied in isolation and purification, and the bioactivity of expression protein was determined in cell proliferation test.MAIN OUTCOME MEASURES: The length and sequence of cDNA segment in hKGF-2, the expression of hKGF-2 gene inE.coli and the purification of hKGF-2 activity.RESULTS: The segment of hKGF-2 cNDA was about 500 bp, and hKGF-2 protein highly expressed in BL-21, which had soluble expression in the supernatant. SDS-PAGE showed that the relative molecular mass was about 20000, and hKGF-2 protein could significantly promote the mitotic activity of NIH3T3 cells. The A value (490 nm) of hKGF-2 in the 1 μg/L, 5 μg/L and 10 μg/L groupswere higher than that in the blank control group, and the differences were significant (which were 0.174±0.022,0.220±0.029,0.306±0.050,0.066±0.004 respectively,P ＜ 0.001).CONCLUSION: hKGF-2 gene is successfully cloned, which highly expresses in BL-21 of the E.coli. Purified hKGF-2 protein can stimulate the proliferation of NIH3T3 cells and significantly promote its mitotic activity.

OBJECTIVEConstructing a plasmid containing the shRNA of luciferase to suppress the expression of luciferase in BHK-21 cell.

METHODSA 334 bp human U6 snRNA promoter was amplified from human genomic DNA by PCR and ligated to a 21 bp reverse repeated motif of luciferase target sequence with 9 bp spacer and AAV plasmid pSNAV. The recombinant pSNAV/U6/Luc plasmid cotransfected with pMAMneoLuc or transfected luciferase cell line to detect the effect of luciferase expression separately.

RESULTSpSNAV/U6/luc suppresses the luciferase expression from pMAMneoLuc by 50% and luciferase cell line by 70%.

CONCLUSIONSThe results showed that the short hairpin RNA of luciferase can efficiently suppress its expression in BHK-21.

Cell Line ; Gene Expression ; Genetic Vectors ; Humans ; Luciferases ; biosynthesis ; genetics ; Plasmids ; genetics ; Promoter Regions, Genetic ; RNA ; genetics ; Transfection

OBJECTIVEStudy on the antiviral effect in vivo and in vitro of recombinant antibodies with neutralization activity to influenza virus HA antigen, IgG-IV-2 and IgG-IV-6 obtained from expression in Baculo/insert system.

METHODSViral titer of influenza virus in MDCK was compared before and after antibody application, and measured viral titer in mouse lung before and after antibodies applied on mucous membrane.

RESULTSIgG-IV-2 and IgG-IV-6 could reduce the titer from 4.5 log10 TCID50 to half is by 50% at doses 0.8 microg and 0.5 microg respectively. When recombinant antibodies were used on mucous membrane, the IgG-IV-2 and IgG-IV-6 reduced the titer by half at doses 0.25 mg/kg weight and 0.1 mg/kg weight respectively. The dose was 0.08 mg/kg weight when the two antibodies were jointly used.

CONCLUSIONSThe recombinant antibodies have antiviral effect in vivo and in vitro, they can neutralize viral virulence.

Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Antigens, Viral ; immunology ; Cell Line ; Influenza A virus ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; Orthomyxoviridae Infections ; prevention & control

As the first class Ⅰ novel genetic engineering medical product in China,recombinant human interferon alpha 1b (IFNα1b) shows unique biological significance and clinical value.Especially in the field of pediatrics,IFNα1 b has been used in the treatment of viral pneumonia,viral hepatitis,bronchiolitis,herpetic angina,hand-footmouth disease and some tumors in children and shown definite efficacy and good safety.It is also used in the prevention and treatment of virus-related wheezing and major infectious diseases,such as influenza.With a greater application prospect,IFNα1b plays an important role in promoting children's health in China.Now,IFNα1b's gene and molecular characteristics,new delivery methods,and the frontier clinical research will be discussed.

BACKGROUNDBased on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.

METHODSTwo unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.

RESULTSThe mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.

CONCLUSIONSThe authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Amino Acid Substitution ; Antibodies, Viral ; Antiviral Agents ; pharmacology ; Cells, Cultured ; DNA Mutational Analysis ; Humans ; Interferon Type I ; genetics ; pharmacology ; Interferon-alpha ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; genetics ; pharmacology ; Plasmids ; genetics ; Receptors, Interferon ; metabolism ; Recombinant Proteins

BACKGROUNDTo construct a series of recombinant herpes simplex viruses that can provide replicating and packaging functions for recombinant adeno-associated virus (rAAV), and to select the strains possessing stronger functions for large-scale production of rAAV.

METHODSA set of cosmids that represents the whole genome of HSV-1 was used to generate recombinant HSV-1 expressing rep and cap proteins of AAV-2. An ATG-to-ACG mutation in the start codon of AAV-2 rep protein was generated by site-directed mutagenesis. rep and cap genes, under control of their native promoters, with or without the ATG-to-ACG mutation in the start codon of rep, were inserted into the Xba site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/ UL2, cos56-rc/ UL44 and cos56-rmc/ UL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1 genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1named HSV1-rc/ UL2, HSV1-rmc/ UL2, HSV1-rc/ UL44 and HSV1-rmc/ UL44 respectively.

RESULTSPCR detection confirmed the existence of rep- gene in the genomes of all four strains of the recombinant HSV-1. Recombinant AAV was produced after infecting the AAV vector cell line that carrying the GFP expression cassette with the four strains of recombinant HSV-1 respectively. However, HSV1-rc/ UL2 and HSV1-rmc/ UL2 produced much more rAAV than HSV1-rc/ UL44 and HSV1c/ UL44 did.

CONCLUSIONSAll the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1 UL2 and HSV1-rmc/ UL2 that provide much stronger functions may be useful for large-scale production of rAAV.

Animals ; Cells, Cultured ; Cosmids ; genetics ; Cricetinae ; Dependovirus ; genetics ; physiology ; Genetic Vectors ; Herpesvirus 1, Human ; genetics ; physiology ; Recombination, Genetic ; Transfection ; Virus Replication