OBJECTIVE: To construct nasopharyngeal carcinoma CNE-2 cell lines expressing stable fusion suicide gene CD/UPRT. UL49. METHOD: The plasmids of pcDNA3.1 (-)E6. BARF1p. CD/UPRT. UL49 was transfected into CNE-2 cells through lipofectamine, and the transfected CNE-2 cells were selected by G418 and prodrugs for getting the cells expressing fusion CD/UPRT. UL49 gene. The protein produced by the suicide gene was tested by Western-blotting in CNE-2 cells. RESULT: Suicide genes were expressed stably in CNE-2 cells. CONCLUSION We constructed nasopharyngeal carcinoma cell lines CNE-2 expressing stable suicide gene through lipofectamine.
Artificial Gene Fusion ; methods ; Carcinoma ; Cell Line, Tumor ; Genes, Transgenic, Suicide ; Humans ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics

Phage display technology refers to a high-throughput in vitro screening technology for extracting required peptides/ proteins from colonies with mass mutants. Due to its high efficiency, practicability and convenience, it has been widely applied in pharmaceutical research and development, as well as target protein screening for active ingredients of traditional Chinese medicines. Target protein is the binding site of drug molecules in vivo, and good targets are the basis of excellent pharmaceuticals. This article summarizes the advance in studies on the phage display technology and its application in targeted protein screening for active ingredients of Chinese materia medica.
Binding Sites ; Cell Surface Display Techniques ; methods ; Drugs, Chinese Herbal ; chemistry ; Mass Screening ; methods ; Plant Extracts ; chemistry ; Proteins ; chemistry

OBJECTIVETo construct dengue virus-specific full-length fully human antibody libraries using mammalian cell surface display technique.

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs) from convalescent patients with dengue fever. The reservoirs of the light chain and heavy chain variable regions (LCκ and VH) of the antibody genes were amplified by RT-PCR and inserted into the vector pDGB-HC-TM separately to construct the light chain and heavy chain libraries. The library DNAs were transfected into CHO cells and the expression of full-length fully human antibodies on the surface of CHO cells was analyzed by flow cytometry.

RESULTSUsing 1.2 µg of the total RNA isolated from the PBMCs as the template, the LCκ and VH were amplified and the full-length fully human antibody mammalian display libraries were constructed. The kappa light chain gene library had a size of 1.45×10(4) and the heavy chain gene library had a size of 1.8×10(5). Sequence analysis showed that 8 out of the 10 light chain clones and 7 out of the 10 heavy chain clones randomly picked up from the constructed libraries contained correct open reading frames. FACS analysis demonstrated that all the 15 clones with correct open reading frames expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces could be detected by FACS analysis with an expressible diversity of the antibody library reaching 1.46×10(9) [(1.45×10(4)×80%)×(1.8×10(5)×70%)].

CONCLUSIONUsing 1.2 µg of total RNA as template, the LCκ and VH full-length fully human antibody libraries against dengue virus have been successfully constructed with an expressible diversity of 10(9).

Animals ; Antibodies, Viral ; CHO Cells ; Cell Surface Display Techniques ; Cricetinae ; Cricetulus ; Dengue Virus ; immunology ; Gene Library ; Genetic Vectors ; Humans ; Immunoglobulin Heavy Chains ; immunology ; Transfection

OBJECTIVETo construct full-length human bladder cancer-specific antibody libraries for efficient display of full-length antibodies on the surface of mammalian cells.

METHODSThe total RNA was isolated from peripheral blood mononuclear cells from patients with bladder cancer. The repertoires of IgG1 heavy chain variable region (VH) and Kappa light chain were amplified by RT-PCR using specific primers. The antibody genes were inserted into the vector pDGB-HC-TM to construct the bladder-cancer-specific antibody libraries of heavy chains and light chains. Ten clones from each library were randomly picked for gene sequencing and transient transfection into FCHO cells to analyze antibody display on mammalian cell surface by flow cytometry after staining with corresponding fluorescent labeled antibodies.

RESULTSThe libraries of bladder-cancer-specific antibody heavy chain (IgG1) and light chain (LCk) were successfully constructed. Seven out of the 10 clones randomly selected from the heavy chain library and 9 out of the 10 clones from the light chain library showed correct open reading frame, coding for 7 unique VH and 9 unique LCk. The combinatory library size reached 3.32×10(11).

CONCLUSIONWe have successfully constructed a full-length human bladder-cancer-specific antibody library with a combinatory diversity of 3.32×10(11) based on mammalian display technology, which can be used for screening monoclonal antibodies against bladder-cancer-associated antigens.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; Cell Surface Display Techniques ; Gene Library ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin kappa-Chains ; genetics ; Peptide Library ; Urinary Bladder Neoplasms ; genetics ; immunology

The gene expressions of codeinone reductase (COR) and berberine bridge enzyme (BBE) in Papaver somniferum were blocked by RNA hairpin of RNA interference (RNAi). The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.
Artificial Gene Fusion ; Genetic Vectors ; NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases ; genetics ; Oxidoreductases, N-Demethylating ; genetics ; Papaver ; enzymology ; genetics ; Plants, Genetically Modified ; enzymology ; genetics ; RNA Interference ; RNA, Small Interfering ; Tobacco ; genetics ; Transformation, Genetic

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

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.
Artificial Gene Fusion ; CD4 Antigens ; biosynthesis ; genetics ; Chemokine CCL20 ; biosynthesis ; genetics ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; physiology ; Humans ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, HIV ; antagonists & inhibitors ; Transfection ; fms-Like Tyrosine Kinase 3 ; biosynthesis ; genetics

We developed a new method for soluble expression of phage-displayed scFv antibody specific for zebrafish vitellogenin. The scFv antibody F5 could bind zebrafish vitellogenin specifically in phage-displayed form but not soluble form. The gene of scFv antibody F5 was cloned into vector pET 32a and transferred into Escherichia coli ori DE3. With inducible expression, soluble scFv antibody 32a-F5 was obtained successfully and could also specifically bind to zebrafish vitellogenin. The insoluble expression of phage-displayed scFv antibody was a common problem in the practical use of phage display. This study offered a feasible way to express soluble scFv antibodies with biological activity.
Amino Acid Sequence ; Animals ; Antibody Specificity ; Base Sequence ; Cell Surface Display Techniques ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Single-Chain Antibodies ; biosynthesis ; genetics ; immunology ; Solubility ; Vitellogenins ; immunology ; Zebrafish ; immunology ; metabolism

OBJECTIVETo construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.

METHODSPeripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry.

RESULTSThe libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10).

CONCLUSIONThe constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.

Amino Acid Sequence ; Animals ; Antibodies ; genetics ; immunology ; Antibody Specificity ; Arthritis, Rheumatoid ; immunology ; Cell Surface Display Techniques ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Immunoglobulin G ; biosynthesis ; genetics ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin kappa-Chains ; biosynthesis ; genetics ; Lymphocytes ; immunology ; metabolism ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.
Animals ; Antigens, Viral ; biosynthesis ; genetics ; Artificial Gene Fusion ; Baculoviridae ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Mice ; Parvoviridae Infections ; prevention & control ; Parvovirus, Porcine ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Somatostatin ; genetics ; Swine ; Vaccines, Virus-Like Particle ; biosynthesis ; immunology ; Viral Vaccines ; biosynthesis ; immunology ; Virion ; genetics ; immunology