Biomedical subjects

Jian-min Li

Publications and source records attributed to Jian-min Li.

10 recordsLinked to original sources

PubMed2007

[Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of anti-human integrin alphanubeta3 monoclonal antibody].

AIM: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection. METHODS: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3. Humanized light chain gene was obtained by screening against human integrin alphanubeta(3). Likewise, humanized Fab were gained by panning human phage antibody library, which was constructed by cloning humanized light chain gene and human heavy chain Fd gene with E10 HCDR3 into pComb3. RESULTS: Three humanized Fab clones was obtained by screening hybrid phage antibody library and human phage antibody library, which contained 2.1x10(6), 2x10(7) colony forming units, respectively. Indirect ELISA and competitive inhibition ELISA analysis demonstrated that three humanized Fab antibody had specific binding activity with human integrin alphanubeta(3). The strongest anti-human integrin alphanubeta(3) reactive D5 clone was sequenced and sequencing analysis showed that the V(kappa) and V(H) were derived from VKIII and VHI, respectively. CONCLUSION: Humanized Fab of anti-human integrin alphanubeta(3) mAb has been successfully obtained by phage display technology which lays the foundation for further clinical research.

Antibodies, Monoclonal↗

PubMed2005

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

AIM: To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. METHODS: The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. RESULTS: A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. CONCLUSION: A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

Amino Acid Sequence↗

PubMed2005

[Study on the coinfection of three tick-borne infectious diseases in China using polymerase chain reaction method].

OBJECTIVE: To study the existence of Ehrluichiosis, lyme disease and tick-borne spotted fever coinfection in some areas in China. METHODS: Using polymerase chain reaction (PCR), B. burgdorferi sensu lato, spotted fever group (SFG) Rickettsiae and human granulocytic ehrlichia (HGE), Ehrlichia chaffeensis (EC) were detected in ticks and mouse samples collected from Inner Mogolia autonomous region, Heilongjiang province, Beijing and Fujian province. RESULTS: 408 Ixodes persulcatus collected from Inner Mogolia autonomous region, HGE and B. burgdorferi sensu lato and SFG Rickettsiae were detected positive, with rates as 6.8%, 7.8%, 45.6%, respectively. 5 (5/408) were coinfection with HGE and B. burgdorferi sensu lato while 1 (1/408) was coinfection with HGE and SFG Rickettsiae. 46 Ixodes persulcatus collected from Helongjiang province were determined positive, with rates as 6.5%, 10.8% and 34.8%, respectively including 1 (1/46) coinfected with HGE and B. burgdorferi sensu lato. 2 of 922 ticks collected from Beijing were detected positive with B. burgdorferi sensu lato. Among 283 groups of Haemaphysalis yeni ticks (3/group) and from 38 rodent samples collected from Ninghua county of Fujian province HCE and B. burgdorferi sensu lato and SFG Rickettsiae were detected. Out of them, 25 groups were positive with EC and the minimal positive rate was 3.8% while 21 rodent samples were positive with EC with a positive rate of 56.4%. 2 ticks and 1 rodent sample were detected positive with EC and spotted fever group. CONCLUSION: Coinfection of HGE and B. burgdorferi sensu lato or spotted fever group Richi did exist in Ixodes persulcatus collected from Inner Mogolia autonomous region and Heilongjiang province. Coinfection of EC and spotted fever group Richi was found in the ticks and rodents collected from Fujian province.

Animals↗

PubMed2005

[The effect of captopril and valsartan on preventing the formation of atherosclerotic plaque].

OBJECTIVE: To observe the effect of captopril and valsartan on preventing the formation of atherosclerosis plaque in rabbit atherosclerotic models. METHODS: The atherosclerotic models of rabbits fed with high-cholesterol diet were used. There were four groups: high cholesterol group, high cholesterol + captopril group, high cholesterol + valsartan group, normal food group. The intima-media thickness in abdominal aortic were measured by ultrasound. The pathogenic features of the arterial walls were showed by HE stain and observed by light microscope. RESULTS: There were less atherosclerotic plaque in high cholesterol + captopril group and high cholesterol + valsartan group than in cholesterol group. The intima-media thickness in high cholesterol + captopril group and in high cholesterol + valsartan group were much more decreased than in high cholesterol group (0.05 +/- 0.005, 0.05 +/- 0.007 vs 0.07 +/- 0.100, P < 0.01). The connection of the endothelial cells were less damaged in high cholesterol + captopril group and high cholesterol + valsartan group than in high cholesterol group. CONCLUSIONS: Ti is true that angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) have the effect of preventing or decreasing the formation of atherosclerotic plaque in arterial wall. The findings suggest that ACEI and/or ARB should be first chosen for those with hypertension and with atherosclerotic risk factors or atherosclerotic diseases.

Angiotensin II Type 1 Receptor Blockers↗

PubMed2005

[The relationship between expression of fas mRNA and apoptosis after traumatic brain injury in rats and the role of GM-1 in protecting the brain].

OBJECTIVE: To explore the mechanism of apoptosis after traumatic brain injury (TBI) in rats and elucidate the role of GM-1 by detecting the expression of Fas mRNA and apoptosis in hippocampi. METHODS: After creating the model of Marmarou cranio-cerebral trauma and offering GM-1 therapy, we observed the expression of Fas mRNA and apoptotic cell death using in situ hybridization and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) technique. RESULTS: Increased expression of Fas mRNA and increased apoptotic cells in hippocampi after TBI were observed. GM-1 could decrease the expression of Fas mRNA and apoptotic cell death. CONCLUSION: The increased expression of Fas mRNA may be a noteworthy cause of apoptotic cell death after traumatic brain injury. GM-1 may play a protective role by way of decreasing the expression of Fas mRNA and apoptotic cell death.

Animals↗

PubMed2005

[Dynamic observation of pathological changes and expression of nuclear factor-kappaB in the intestinal mucosa of rats after diffuse brain injury].

OBJECTIVE: To investigate the dynamic pathological changes in the intestinal mucosa of rats and the role of activation of nuclear factor-kappaB (NF-kappaB) in the alteration in intestinal mucosal barrier dysfunction after diffuse brain injury. METHODS: The animal model established by Marmarou was used to produce diffuse brain injury. One hundred and fifty male Wistar rats were randomly divided into nine groups in terms of postinjury time: 1, 2, 4, 8, 12, 24, 48, 72, 168 hours after injury, and control group. Mucosal thickness, villous height and width were estimated under light microscope, and expression of NF-kappaB was assessed with immunohistochemical staining in control group to compare with those in injury groups. RESULTS: Mucosal thickness, villous height and width were diminished and the optical density of expression of NF-kappaB was much higher in the injury groups than those of control group. CONCLUSION: The structure of intestinal mucosa is damaged in the earlier stages after diffuse brain injury. NF-kappaB is activated by cellular stress, which may be involved in the pathogenesis of secondary intestinal lesion.

Animals↗

PubMed2004

[The initiation of G2/M checkpoint by diallyl disulfide requires the activation of p38 MAP kinase in HL-60 cells].

OBJECTIVE: To explore the molecular mechanisms of G(2)/M checkpoint initiated by diallyl disulfide (DADS) in HL-60 cells. METHODS: Cell viability was determined by MTT assay. Cell cycle was assayed by flow cytometry. The expression of phospho-p38, Cdc25B and Cdc2, and p38 mRNA were measured by Western blotting and RT-PCR, respectively. RESULTS: After treatment with DADS at 5 - 160 micro mol/L for 0 - 72 h, the growth of HL-60 cells were suppressed in a concentration-dependent manner and the inhibitory effect of DADS (20 micro mol/L) was similar to that of ATRA (10 nmol/L) (P > 0.05). Incubation of HL-60 cells with DADS (20 micro mol/L) for 12 h could activate G(2)/M checkpoint and increase the expression of phospho-p38 MAPK, followed by the expression of phospho-Cdc25B and phospho-Cdc2 (P < 0.05). SB202190, a specific inhibitor of p38 MAPK, markedly blocked the phosphorylation of p38 MAPK, Cdc25B and Cdc2 (P < 0.05). CONCLUSION: DADS could induce the G(2)/M arrest in HL-60 cells which may be involved in the activation of p38 MAP kinase.

Allyl Compounds↗

PubMed2004

[Construction and expression of anti-human integrin alphavbeta3 scFv].

AIM: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3. METHODS: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E. coli BL21 (DE3). RESULTS: SDS-PAGE analysis showed the expressed recombinant protein with relative molecular mass (Mr) being 31,000. Western blot confirmed that the protein was labeled with His6. scFv protein was expressed as soluble protein under the condition of a small amount of IPTG induction and culture at lower temperature. The purity of the protein purified through Ni-NTA agarose metal affinity resin column was over 91%. The purified protein could bind to the human integrin alphavbeta3 by ELISA confirmation. CONCLUSION: scFv against human integrin alphavbeta3 has been successfully constructed and expressed,which lays the foundation for further clinical research.

Amino Acid Sequence↗

PubMed2004

[Construction of eukaryotic vector and expression of chimeric antibody against rh-bFGF].

AIM: To express a human-mouse chimeric antibody against rh-bFGF antigen in eukaryotic cells. METHODS: The VL and VH genes were amplified and cloned from the hybridoma 1F11 secreting anti-rh-bFGF mouse monoclonal antibody (mAb) and the CL and CH genes were amplified and cloned from the plasmid pMDHC and pMDLC. They were inserted into eukaryotic expression vectors after sequencing. The recombinant plasmids were then transformed into CHO-dhfr- cells for expression. The humanization and antigen specificity of expressed products were identified by indirect ELISA. The relative molecular mass (Mr) of expressed products was determined by SDS-PAGE. RESULTS: The cloned antibody genes were identified to be functional by sequencing. The chimeric antibody could be detected in the culture supernatant of transformed CHO cells. The humanization and specificity of the chimeric antibody to rh-bFGF were confirmed by ELISA. CONCLUSION: The mouse-human chimeric antibody against rh-bFGF has been expressed successfully in eukaryotic cells, which lays the foundation for its further clinical research.

Amino Acid Sequence↗

PubMed2002

[Endothelin-1 expression and quantitative analysis in astrocytomas].

BACKGROUND & OBJECTIVE: It is demonstrated that endothelin-1 (ET-1) is synthesized by and released from certain malignant tumor cells, and it plays an important role in growth and development of tumor. This study was designed to investigate the expression of ET-1 in astrocytomas and the relationship between the ET-1 quantity and the grades of astrocytomas. METHOD: ET-1 expression was determined in 70 astrocytoma specimens using Streptavidin-Peroxidase method and image analysis technology. RESULTS: The ET-1 expressed in all of astrocytomas, and the ET-1 expression was mainly located in the cytoplasm. The positive rates of ET-1 in grade IV and III astrocytomas (86.67% and 93.33%) were significantly higher than that in grade II, I and normal astrocytes(75.00%, 66.67%, and 37.50%) (P < 0.05). The results of image analysis on astrocytoma (grade IV, III, II, I and normal control: 0.1875 +/- 0.0227, 0.1516 +/- 0.0134, 0.1215 +/- 0.0116, 0.1048 +/- 0.0143, and 0.0717 +/- 0.0074, respectively) showed that the lower differentiation of astrocytoma, the higher ET-1 expression (P < 0.01). The expression of ET-1 was significantly correleted with the tumor grading (r = 0.863, P < 0.01). CONCLUSION: The ET-1 quantitative analysis may be used as a monitoring index of astrocytoma growing.

Adolescent↗