Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.
Animals ; Immunoglobulin Variable Region/genetics* ; Immunoglobulin Fragments/metabolism* ; Single-Chain Antibodies/metabolism* ; Peptide Library ; Mammals/genetics*

BACKGROUNDThe large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies.

METHODSscFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated.

RESULTSThe phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression.

CONCLUSIONSThe reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

Bacteriophages ; genetics ; Digoxin ; immunology ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Immunoglobulin Fragments ; biosynthesis ; immunology ; Peptide Library

Tissue factor pathway inhibitor (TFPI) is one of the major physiological inhibitors of the human blood coagulation cascade and may have great potential in the prevention and therapy of diseases caused by thrombus formation. In this study, recombinant human tissue factor was generated in E. coli containing a recombinant vector constructed by inserting TFPI cDNA into pGEX-2T vector. The generated recombinant TFPI (rTFPI) could be simply purified with glutathione-agarose affinity method and maintained its biological function in terms of inhibition of tissue factor and factor Xa.
Escherichia coli ; genetics ; metabolism ; Humans ; Lipoproteins ; biosynthesis ; genetics ; pharmacology ; Peptide Fragments ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology ; Transfection

OBJECTIVETo explore the effect of human endostatin expressed by host cells on the growth of human liver carcinoma in vivo.

METHODSHuman endostain gene was transferred into SMMC7721 cells by retroviral pLncx to build endostatin-transfected cell line. PCR, immunohistochemistry and Western blot analysis were applied to examine the transfection, expression and secretion of endostatin. Endothelial cell proliferation assay was used to determine the biological activity of expressed endostatin. The in vivo and in vitro growth rates of the endostatin-transfected and control SMMC7721 cells were also observed.

RESULTSPCR proved that the genome of endostatin-transfected SMMC7721 cells contained a 550 bp specific fragment of endostatin. The expression and secretion of human endostatin from endostatin-transfected SMMC7721 cells were confirmed by immunohistochemistry and Western blot analysis. Endostatin expressed by host cells could inhibit the proliferation of human umbilical vein endothelial cells by 48% (P < 0.01). In vitro proliferation assay showed that endostatin-transfected SMMC7721 cells had no change in proliferation rate compared to control SMMC7721 cells. In comparison with control group, however, tumor growth rate in vivo from endostatin-transfected SMMC7721 cells was inhibited greatly by 94.5%, 22 days after inoculation into nude mice (P < 0.01).

CONCLUSIONHuman endostatin mediated by retroviral gene transfer can inhibit greatly the growth of human liver carcinoma SMMC7721 in vivo.

Animals ; Carcinoma, Hepatocellular ; therapy ; Collagen ; genetics ; Endostatins ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Mice ; Peptide Fragments ; genetics ; Retroviridae ; genetics ; Transfection

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.
Amyloid beta-Peptides ; genetics ; immunology ; Antibody Specificity ; immunology ; Humans ; Peptide Fragments ; genetics ; immunology ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification

Heat shock protein gp96 is a glycoprotein which was found several years ago. Besides its function as a molecular chaperone, it is also reported to play important roles both in innate immunity and adaptive immunity. Gp96 can stimulate the maturation of antigen presenting cells (especially dendritic cells) and the secretion of cytokines. Gp96 and its associated peptides could stimulate peptide specific cytotoxic T lymphocyte reaction (CTL), which was very promising in the designing of anti-virus and anti-tumor vaccines. However the expression level of whole length gp96 was relatively low in E. coli and the purity of gp96 are not very suitable for further study. We successfully cloned the carboxy terminal fragment (560aa-751aa) of murine gp96 into the pGEX-6p-1 vector and expressed in BL21 strain. This fragment contains the peptide binding domain and the dimerization domain. After purification, the recombinant fusion protein was cleaved with the PreScission Protease and analyzed by Gelfiltration. The results show that this fragment may be related to the dimerization of gp96 and make an foundation for further investigations of the protein.
Animals ; Antigens, Neoplasm ; genetics ; Blotting, Western ; Chromatography, Gel ; Cloning, Molecular ; Mice ; Peptide Fragments ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; isolation & purification

OBJECTIVE: To explore the clinical characteristics and variants of ATP7A gene in a child with Menkes disease. METHODS: A child with Menkes disease diagnosed at the West China Second Hospital of Sichuan University and its family members in March 2022 was selected as the study subjects. Clinical manifestations and results of laboratory tests and genetic testing were summarized. RESULTS: The main manifestations of the child included seizures, global development delay, facial dysmorphism, sparse and curly hair, increased lactate and pyruvate, and significantly decreased cuprin. EEG showed frequent issuance of multifocal spikes, spines, polyspines (slow) and polymorphic slow waves. Multiple tortuous vascular shadows were observed on cranial MRI. Whole exome sequencing revealed that the child has harbored a hemizygous c.3076delA (p.ile1026*) variant of the ATP7A gene, which was inherited from his mother. The variant may lead to premature termination of protein translation. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted as pathogenic (PVS1+PM2+PP4). CONCLUSION The c.3076delA (p.Ile1026*) variant of the ATP7A gene probably underlay the Menkes disease in this child. Above finding has provided evidence for clinical diagnosis. The significantly increased lactic acid and pyruvate can be used as a reference for the diagnosis and management of Menkes disease. Microscopic abnormalities in the hair of the carriers may also facilitate their diagnosis.
Child ; Humans ; Copper-Transporting ATPases/genetics* ; East Asian People ; Menkes Kinky Hair Syndrome/genetics* ; Mutation ; Pedigree ; Peptide Fragments ; Pyruvic Acid

OBJECTIVETo obtain recombinant fusion protein HSA (human serum albumin)-PTH(1-34) in Pichia pastoris.

METHODSHSA and PTH(1-34) cDNA were obtained with PCR and the DNA segments were cloned into vector pPIC9 with linker. The linearized plasmids were transformed GS115 competent cells treated with LiCl, and mut+ transformants were screened on MD plate. With AOX promoter and alpha-MF signal sequences leading, fusion protein was expressed in GS115. PCR and SDS-PAGE were employed to confirm the integration and expression of HSA-PTH(1-34). The fusion protein was identified by Western blotting and classical adenylate cyclase assay.

RESULTThe PCR results showed that the gene of HSA-PTH(1-34) was integrated into GS115 genome. Western bolt approved the existence of two domains of HSA and PTH(1-34). The bioactivity assay in rabbit cortical membranes indicated that HSA-PTH (1-34) activated adenylate cyclase, but the activity was lower than that of the synthetic PTH(1-34).

CONCLUSIONActive fusion protein HSA-PTH (1-34) is successfully expressed in Pichia pastoris.

Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Peptide Fragments ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics ; Teriparatide ; analogs & derivatives

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.
Adenoviridae ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Genetic Vectors ; genetics ; Humans ; Neovascularization, Physiologic ; genetics ; physiology ; Peptide Fragments ; genetics ; physiology ; Plasminogen ; genetics ; physiology ; Polymerase Chain Reaction

OBJECTIVETo construct a human phage display antibody library, which will help to develop new drugs and vaccines against respiratory syncytial virus (RSV) and solve many of the issues that have limited the progression and application of murine monoclonal antibodies (McAbs) in the clinic. This can provide a platform for human antibody preparation and diagnosis, prophylaxis and therapy of RSV infection in children.

METHODSPeripheral blood lymphocytes were isolated from 52 children with RSV infection. cDNA was synthesized from the total RNA of lymphocytes. The light and heavy chain Fd (VH-CH1) fragments of immunoglobulin gene were amplified by RT-PCR. The amplified products were cloned into phagemid vector pComb3x and the clone samples were electrotransformed into competent E.coli XL1-Blue. The transformed cells were then infected with M13K07 helper phage to yield recombinant phage antibody of Fabs. The plasmids extracted from amplified E.coli were digested with restriction endonucleases Sac I, Xba I, Spe I and Xho I to monitor the insertion of the light or heavy chain Fd genes. RSV virions were utilized as antigens to screen Fab antibodies.

RESULTSBy recombination of light and heavy chain genes, an immune Fab phage display antibody library against RSV containing 2.08x10(7) different clones was constructed, in which 70% clones had light chains and heavy chain Fd genes. The capacity of Fab phage antibody gene library was 1.46x10(7) and the titre of the original Fab antibody library was about 1.06x10(12) pfu/mL. The antibody library gained an enrichment in different degrees after the preliminary panning.

CONCLUSIONSUtilizing the technology of phage display, an immune Fab phage display antibody library against RSV was successfully constructed in this study, which laid a valuable experimental foundation for further study and created favorable conditions for preparing human McAbs. This may also contribute to the improvement in the diagnosis, therapy and prophylaxis of RSV infection in children.

Antibodies, Viral ; genetics ; Bacteriophages ; genetics ; Child ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Peptide Library ; Respiratory Syncytial Viruses ; immunology