Reconstruction and Analysis of A Human Small Molecular Antibody to Tumor Necrosis Factor Alpha.

A Fab antibody gene was constructed on the basis of the reconstruction of the linker of a human anti-TNF-alpha ScFv gene. The two ScFvs before and after reconstruction were cloned into expression vector pBV220. About 30 kD recombinant proteins were expressed by induction and they constituted 6.5% and 13.8% of the total bacterial protein, respectively. A soluble Fab expression vector was constructed and transformed into E.coli HB2151.After induction by IPTG, a new protein band about 50 kD appeared on SDS-PAGE. The expressed ScFv and Fab were purified from E.coli lysates, and further experiments showed that 1) the expression amount of reconstructed ScFv was increased distinctly 2) ScFv and Fab could bind rhTNF-alpha. The ScFv containing GGGGS had an affinity constant of 6.70x10(4)(mol/L)(-1), and the ScFv containing (GGGGS) (3) had an affinity constant of 7.27x10(5) (mol/L) (-1).The affinity constant of Fab was 7.61x10(5) (mol/L) (-1). The Fab and reconstructed ScFv was indifferent in affinity activity 3) ScFv and Fab neutralized the cytotoxicity of rhTNF-alpha. The neutralizing ability of Fab was the same as the reconstructed ScFv, but lower than a mouse anti-TNF-alpha mAb. These data may be helpful for using human anti-TNF-alpha small molecular Ab in antagonizing the activity of TNF-alpha in therapy.

Identification of the Peptides that Bind to Tyrosine Kinase Receptor EphB2 by Phage Display.

The gene encoding the Ig-like domain of tyrosine protein kinase receptor EphB2 was cloned into the expressing vector pET28a. Under induction with IPTG, the positive strain expressed the fusion protein with a hexahistidine tail on the N-terminal. The protein was purified under denaturing conditions using metal chelate chromatography. The purity was up to 94%. The purified-protein-coated ELISA plate was used as target to screen recombinant phages able to bind onto it, and after three rounds of affinity screening, 19 phages that could bind specifically with EphB2 were isolated from a random phage-displayed seven-peptide library. The peptide sequences of the positive phage clones were analyzed.

Mechanism of exogenous nucleic acids and their precursors improving the repair of intestinal epithelium after gamma-irradiation in mice.

AIM:To clone expressed genes associated with repair of irradiation-damaged mice intestinal gland cells treated by small intestinal RNA, and to explore the molecular mechanism of exogenous nucleic acids improving repair of intestinal crypt.METHODS:The animal mode of test group and control group was established, forty-five mice being irradiated by gamma ray were treated with small intestinal RNA as test group, forty mice being irradiated by gamma ray were treated with physiological saline as control group,five mice without irradiation were used as normal control, their jejunal specimens were collected respectively at 6h, 12h,24h, 4d and 8d after irradiation. Then by using LD-PCR based on subtractive hybridization, these gene fragments differentially expressed between test group and control group were obtained, and then were cloned into T vectors as well as being sequenced. Obtained sequences were screened against. GeneBank, if being new sequences, they were submitted to GeneBank.RESULTS:Ninety clones were associated with repair of irradiation-damaged intestinal gland cells treated by intestinal RNA. These clones from test group of 6h, 12h, 24h, 4d and 8d were respectively 18, 22, 25, 13, 12. By screening against GeneBank, 18 of which were new sequences, the others were dramatically similar to the known sequences, mainly similar to hsp, Nmi,Dutt1, alkaline phosphatase, homeobox, anti-CEA ScFv antibody, arginine/serine kinase and BMP-4,repA. Eighteen gene fragments were new sequences,their accept numbers in GeneBank were respectively AF240164-AF240181.CONCLUSION:Ninety clones were obtained to be associated with repair of irradiation damaged mice intestinal gland cells treated by small intestinal RNA, which may be related to abnormal expression of genes and matched proteins of hsp, Nmi, Dutt1, Na, K-ATPase,alkalineph-osphatase, glkA, single stranded replicative centromeric gene as well as 18 new sequences.

Binding of Insulin Receptor Substrate 1 PH Domain to Protein Kinase C.

To screen for novel ligands of insulin receptor substrate 1(IRS-1)PH domains, to investigate the role of the PH domains in signal transduction processes and to examine association of the PH domain with protein kinase C(PKC), rat liver was employed to clone the gene encoding for the PH domains. Total RNAs were isolated and purified from fresh liver and mRNAs were reversely transcribed into cDNAs. After PCR the fragments of the DNA were cloned into vector pUC19. The sequence of the fusion gene was confirmed by sequencing, and the gene was correctly expressed in E.coli as fusion protein with glutathione S- transferase(GST). The fusion protein was purified by glutathione agarose beads, then was incubated with the lysate of Jurkat cells. After SDS-PAGE, the proteins were transferred to PVDF membrane, and an anti-PKC antibody was used to detect binding between the PH domain with PKC. The sequence of the gene encoding for the PH domains was confirmed to be correct, and the PH domain was successfully expressed in E.coli JM 109 in soluble form. Western blots confirmed the binding of the PH domain with PKC in vitro. In conclusion, purified IRS-1 PH domain GST fusion protein was obtained and its biological activity was confirmed.

Expression of the Variable Region Genes of the Monoclonal Antibodies Against Metal-bound Tetrapeptide in E. coli.

The variable region genes of the light and heavy chains obtained from three stems of McAb against metal-bound tetrapeptide were joined into a single chain by a linker. A 39 bp fragment of the N-terminal of CGRP was joined to the C-terminal of the heavy chain to constitute the Lv-linker-Hv-CGRP single chain gene which was cloned into the vector pTC01 and expressed in E. coli 71/18. The molecular weight of the expressed product was approximately 26 kD as shown by SDS-PAGE. Its expression level was about 20%-30% to he total cellular proteins. The product was a soluble protein and showed binding activity with its hapten by indirect ELISA assay.