Biomedical subjects

Qing-Jun Ma

Publications and source records attributed to Qing-Jun Ma.

15 recordsLinked to original sources

PubMed2006

[Expression in Pichia pastoris and properties of human serum albumin-interferon alpha2b chimera].

To reduce the serum clearance of interferon alpha2b, a chimeric gene encoding an human serum albumin(HSA)--human interferon alpha2b(IFNalpha2b) fusion protein was overexpressed in Pichia pastoris. After fermentation in a 5L bioreactor, the fusion protein, capable of cross-reacting with anti-IFN alpha and anti-HSA antibody, was purified from the culture of the recombinant yeast by ultrafiltration, blue Sepharose affinity, phenyl hydrophobic interaction and Q ion exchange chromatography. Its IFNa2b moiety exhibits antiviral activity similar to that of recombinant human IFNa2b. In Cynomolgus monkeys model, The fusion protein was detectable in plasma, even 336h after a single does of 90 microg/kg injection intravenously or subcutaneously. The elimination phase half-life of the fusion protein was 101h after intravenous injection and 68.2h after subcutaneous injection. Its Subcutaneous bioavailability was 67.9%. The enhanced pharmacokinetics of interferon a2b fused to human serum albumin suggest its promissing application in clinic medicine.

Animals↗

PubMed2006

[SCT-OGP fusion protein expression and its potential role as osteoporosis therapy].

Osteoporosis is a disease of bone metabolic disorder, the incidence of which increases sharply in old people. Calcitonin (CT) is a peptide hormone containing 32 amino acid that can inhibit osteoclasts activity. Osteogenic Growth Peptide (OGP) is a peptide hormone with 14 amino acid. It is an autocrine mitogen for osteoblastic and firbroblastic cells which has anabolic activiy. Six SCT and OGP DNA segments were chemically synthesized and ligated into a yeast expression vector pPIC9. The recombinant plasmid was transformed into pichia pastoris GS115. Finally, we got two stable SCT-OGP high expression clones after screening. Purifed protein can stimulate the proliferation of osteoblastic and fibroblastic cells, and also stimulate serum ALP activity and decrease serum calcium level using mice as animal models, demonstrating its potential role in oteoporosis therapy.

Animals↗

PubMed2005

[Surgical factors underlying the recurrence of primary spine tumor].

OBJECTIVE: To find the causes of the spinal primary tumors recurrence in surgical technique. METHODS: From 1989 to 2002, 38 cases of primary spine tumors, including giant cell tumor, osteoblastoma, chondrosarcoma and chordoma with wide resection via a piece-meal fashion. By adopting a retrospective method, the present study investigated the clinical and imaging materials of pre- and post-operation period and those in follow-up. RESULTS: The study included 18 cases of giant cell tumor, 6 osteoblastoma, 6 chondrosarcoma and 8 chordoma. In all cases, 63% of tumors were in cervical and cervicothoracic (C(7)-T(2)) spine; 29% in thoracolumbar (T(4)-L(5)) spine and 8% in sacrum. Tumors involved in multi-segment were 34%. And 71% patients had the tumor in the body and the arch simultaneously. And 71% of tumors formed paraspinal masses, 42% in both sides. The tumors invaded the channel in 58% of the cases. The compartment were invaded in 79% patients. Finally, 32 patients were followed up, from 1.0 approximately 14.9 years, 5.1 years average. Seventeen patients recurred after the surgery, the recurrence rate was 53%. The recurrence rate of giant cell tumor was 35%, osteoblastoma 50%, chondrosarcoma 75%, chordoma 100%. The recurrence rate of tumor in cervical and cervicothoracic spine was 63%, thoracic-lumbar 33%, sacrum 67%. The recurrence rate of multi-segment tumors was 80%, and that of single segment 41%. The recurrence rate of the tumors involving in vertebral body alone or involving the arch simultaneously reached 75% and 55% respectively; the recurrent rate in the arch alone was 33%. The recurrence rate of the tumors involving in vertebral body ranged in different segments. Those growing in cervical and cervicothoracic spine reached 73%; those growing in thoracolumbar spine was only 25%. The recurrence of the tumors without soft masses was 20%, those of single-sided soft masses was 45% and those of double-sided 91%. Among the 17 recurrent patients, 83% of the tumors were in the cervical and cervicothoracic spine. Those extending to the upper cervical and cervicothoracic amounted to 58%. All the 17 recurrent patients had body lesion and paraspinal soft masses. CONCLUSIONS: During the primary spinal tumor operation, that failure to get adequate exposure and full division is thought to be the cause of recurrence. So the precise design before surgery and adequate exposure of the tumor in the surgery is the guarantee of wide excision.

Adolescent↗

PubMed2005

[Enhancement of immune responses to hepatitis B DNA vaccine by superantigen SEA in mice].

To investigate the adjuvant effect of plasmid DNA encoding superantigen SEA (D227A) (pmSEA) on immune responses induced by HBV DNA vaccine containing HBV preS2 and S antigen in BABL/c (H-2d). BALB/c mice were immunized intramuscular injection with HBV DNA vaccine (pHBVS2S) mixed with or without pmSEA plasmid. Antibodies againat HBV PreS2 and S antigen in the sera were accessed by Anti-HBs ELISA, and the HBsAg specific cytotoxic T lymphocytes (CTLs) activity was determined by 5 Chromium Release Assay. The HBs peptide-specific IFN-gamma secreting T cells were detected by ELISPOT. Anti-HBs antibody titers and CTLs activity in mice immunized with pmSEA + pHBVS2S group were significant higher (P < 0.05) than pHBVS2S DNA vaccine group. The ratio of IgG1/IgG2a (0.282) was apparently different from the group immunized with peptide (10). Mice immunized with HBV DNA vaccine plus adjuvant produce higher titer of IgG1 and IgG2a antibodies against HBV S antigen 1.36 and 1.73 time higher than that without adjuvant respectively. HBs peptide--specific IFN-gamma secreting T cells increased 2 - 3 times by the pmSEA adjuvant, compared to DNA vaccine group. HBV DNA vaccine (pHBVS2S) induces humoral and cellular immuno-responses in BALB/c mice, and the responses could be significantly boasted by the plasmid encoding mSEA. Therefore the pmSEA was a potential adjuvant for DNA vaccines.

Adjuvants, Immunologic↗

PubMed2004

[Induction of protective immunity in rhesus monkey by inoculation with recombinant fusion protein of cholera toxin B subunit-multivalent epitopes of Plasmodium falciparum].

Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.

Animals↗

PubMed2004

[Functional expression of DNA binding domain of Zif268 in Escherichia coli].

As the ubiquitous nucleic acids recognizing motif, Zinc finger protein play important role in regulation of gene expression. The study of recognization specific will greatly facilitate understanding the delicate interaction of Zinc finger protein and DNA. By the choice of expression vector, the induction and culture conditions, the DNA binding domain of Zif268 was expressed in Escherichia coli partly solubly. The gel mobility shift assay shows that purified DNA binding domain can bind its natural target sequence specifically, which indicates the DNA binding domain remains its DNA binding activity in Escherichia coli. The functional expression of DNA binding domain of Zif268 will greatly facilitate the development of in vivo genetic selection assay for the study of Zinc fingers-DNA interaction.

DNA-Binding Proteins↗

PubMed2003

Crystal structure of beta-luffin, a ribosome-inactivating protein, at 2.0 A resolution.

The crystal structure of beta-luffin at 2.0 A resolution was solved by the molecular-replacement method using polyalanyl trichosanthin as the search model. The structure was refined with CNS1.1, giving R(work) = 0.162 and R(free) = 0.204. The r.m.s.d.s of the bond lengths and bond angles are 0.008 A and 1.3 degrees, respectively. The overall structure is similar to those of other type I RIPs. Three N-acetylglucosamine (Nag) molecules are linked to residues Asn2, Asn78 and Asn85 of the protein.

Asparagine↗

PubMed2003

[Study on relationship between length of homologous sequences and chromosomic recombination rate in Saccharopolyspora erythraea].

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.

Chromosomes, Bacterial↗

PubMed2003

[High expression and characterization of human parathyroid hormone in Escherichia coli].

Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.

Amino Acid Sequence↗

PubMed2003

[Over-expression in Escherichia coli and purification of nucleocaspid and membrane protein of SARS coronavirus].

Genes encoding nucleocaspid (N) and membrane (M) protein of SARS coronavirus were obtained by RT-PCR and were cloned into expression vector pET22b and pBV222. DNA sequencing showed that the genes cloned from a patient in Beijing were identical to the gene sequences from reported Toronto strain. The genes were over-expressed in E. coli either as inclusion body or as soluble form. The recombinant proteins were purified by ion-exchange, or ion-exchange followed by metal chelate affinity chromatography. The recombinant N protein was demonstrated highly antigenic and could be employed as antigen to detect SARS antibodies in ELISA system for SARS diagnosis.

Chromatography, Affinity↗

PubMed2003

[Gene cloning, soluble expression and activity analysis of rSEA].

The superantigen,such as staphylococcal enterotoxins, had been identified as possible anti-cancer molecules in many reports. In this paper, we cloned the entA gene encoding Staphylococcal enterotoxin A from the genomic DNA of Staphylococcus aureus(ATCC13565) by PCR, the sequence cloned was accordance with that reported in Genebank. The entA gene could be expressed effectively after inserted into plasmid pET-22b( + ), The rSEA was expressed as inclusion bodies when induced by IPTG at 37 degrees C and became soluble after induced at low temperature, the soluble part is about 55% of total rSEA products. Only one band was detected by western-blotting in expression product of BL-21 (DE3) with pET-SEA. The soluble rSEA was purified by Ni2+ chelating sepharose column. No other protein except rSEA was seen in SDS-PAGE gel stained by both Coomassie brilliant blue and silver salt, which showed that the rSEA was purified effectively. Homology modeling of rSEA determined the structure change was conducted, which indicated there was no apparent structure change between rSEA and native SEA. This result was also confirmed by proliferation assay of PBMC, for the rSEA could induced proliferation of PBMC as effectively as native SEA. The increasing anti-tumor activity of rSEA was also detected after the spleen cell activated in vivo by rSEA, which was accordance with others reports. This work paved the way for the further study of anti-cancer with rSEA.

Cell Line, Tumor↗

PubMed2003

[Expression plasmid-host strain using chromosome-plasmid balanced lethal system based on the Escherichia coli thyA].

To construct a vector for DNA vaccine and protein expression by using chromosome-plasmid balanced lethal system which was based on the thyA+ gene/deltathyA Escherichia coli. The thyA genes from Escherichia coli and Vibrio cholerae were amplified by polymerase chain reaction and cloned into pCDNA3 by replacing ampilicilin resistant gene. Multiple cloning sites, the prokaryotic replicon, CMV promoter and the boving growth hormone polyA signal were also included in the vectors. Two new non-antibiotic recombinant plasmids renamed as pcDNATE and pcDNATC which had the nutritional marker as thyA were constructed and were transformed respectively into the deltathyA derivative of E. coli K-12 strain DY330-TI, then two chromosome-plasmid balanced systems for E. coli based on the thyA were developed. To test the efficiency and stability of the newly constructed chromosome-plasmid balanced lethal system, a reporter gene--red fluorescent protein (DsRed2) gene was cloned into pcDNATE, pcDNATC and expressed as fusion to the c-myc. The two recombinant plasmids, pcDNATE-DsRed2, pcDNATC-DsRed2, were transfected into HEK293 solely and DsRed2-myc was detected by the fluorescence microscope assay and western-blot. Meanwhile, the loss of recombinant plasmids were not seen in cultures without thymidine after 20 generations. The chromosomal-plasmid balanced lethal system is proved to be an effective vector system for the expression of target genes and share the same stability with the antibiotic-resistant plasmid vector system. It holds great potential in gene vaccine vector because obviating the weakpoints of the drug resistance marker during application.

Bacterial Proteins↗

PubMed2003

[Prophylactic and therapeutic vaccines against Alzheimer's disease].

Beta-amyloid(Abeta) immunization as vaccines has now become a promising approach for the prevention and treatment of Alzheimer's disease (AD)after its debut in 1999. Transgenic mouse models of AD that develop age-dependent Abeta deposition, damage to the neuropil, and behavioral deficits have enabled researchers to test if the approach can influence these AD-like pathologic changes in their brains. Active immunization with different forms of A beta and protocols have been shown to decrease brain Abeta deposition and improve cognitive performance in these mice models in the following studies. Although the phase II clinical trials of active immunization with Abeta(AN1792)were halted last year due to the occurrence of CNS inflammation in a small subset of patients, researchers found that strong humoral responses can be induced by the vaccination. Furthermore, the active immunization also brings an almost complete clearance of Abeta from much of the cerebral cortex. Abeta specific antibodies are believed to cross blood-brain barrier by minimal destroy of vascular wall where amyloid depositions exist. Three possible mechanisms on removal of Abeta deposition from brain have also been reviewed. Still some problems should be clarified before this strategy could be applied for clinical therapy. Whether vaccination will improve the cognitive decline in AD patients will depend upon clinical assessments, which was vital to destiny of the approach.

Alzheimer Disease↗

PubMed2003

[Study of recombinant stem cell factor].

Stem cell factor is an important hematopoietic growth factor. In this study, the human stem cell factor was produced by recombinant E. coli, and the structure and biological activity of the recombinant stem cell factor(rhSCF) was studied. It was indicated that the rhSCF was a uncovalent dimer in phosphate buffer,and had the correct mass spectra, mass peptides spectra, composition of amino acid, N-terminal sequernce, C-terminal sequence and intrachain disulfide linkages, rhSCF alone or synergy with rhG-CSF could mobilze hematopoietic progenitors to blood in monkey.

Animals↗

PubMed2002

[Construction of Saccharopolyspora erythraea M synthesizing a novel ketolide 3-deoxy-3-oxo-erythronolide B].

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.

Anti-Bacterial Agents↗