Objective: To fuse Mycobacterium tuberculosis protective antigen gene with mice ubiquitin gene, constructing antigen ubiquitin system. Methods: Mice ubiquitin cDNA was amplified by RT PCR from mice testicle,and 4 antigen genes were obtained by PCR from cultured Mycobacterium tuberculosis . Ubiquitin and 4 antigen genes were linked by flexible adaptor respectively and the fusing genes were cloned into pVAX vector.The recombinant plasmids were digested with endonuclease and sequenced.Then the recombinant plasmids were transfected into COS7 cells and the expression was assayed by ELISA. Results:Ubiquitin and 4 antigen genes were 0.2,0.3,0.7,1.0,1.65 kb in length by agarose electrophoresis. Endonuclease digestion of the recombinant plasmids indicated that the fusion genes were correctly inserted into pVAX vector. Sequencing results of fusion nucleic acid vaccines were identical to those in GenBank.The recombinant plasmids expressed in COS7 cells. Conclusion: Four Chinese Mycobacterium tuberculosis protective antigens ubiquitin systems are successfully constructed and can be expressed in eukaryotic cells. This may provide a basis for diagnosis and therapy of tuberculosis.

Objective To analyze the clinical features and the mutation of LEMD3 gene in four osteopoikilosis patients.Methods Clinical data of 4 patients were collected, peripheral blood samples were obtained for DNA extract, and LEMD3 gene mutation was analyzed by direct DNA sequencing.Results 4 patients with osteopoikilosis included a male aged 44, a female aged 42, a 26-year-old male, a 21-year-old female.All these patients were from families of non-consanguineous marriage.The main complaint of these patients was pain on arthrosis.Abnormal X-ray radiography was found in medical examination, while markers of bone metabolism were normal.The results of X-ray examination showed that numerous, discrete round or ovoid calcification were scattered throughout the wrist, pelvis and scapula.A de novo mutation c.595delG(NM_014319.4) localized in exon1 of the LEMD3 gene resulting in p.Ala199ProfsX46 of Case 3, while the mutation is not found in his parents and the remaining 3 patients.Conclusions A de novol LEMD3 mutation led to osteopoikilosis was found, and the pathogenesis of molecular mechanism in Chinese remained further exploration.

Objective: To screen out DNA binding proteins specifically recognizing CpG immunostimulatory sequence (ISS) for further investigating the molecular mechanisms of ISS. Methods: Yeast one hybrid system was adapted in screening a human bone marrow cDNA library using 4 copies of ISS as bait. The ISS binding activity of the positive clone was confirmed by electrophoretic mobility shift assay (EMSA). Results: Four dual positive colonies were obtained, two of them encoded proteins with unknown functions. The other 2 encoded light chains of immunoglobulin with amino sequences homology to anti DNA Ab and HBsAb respectively. EMSA showed HBsAb specifically bound to CpG ODN at pH6.4 and pH 5.8. Conclusion: HBsAb may have ISS specific DNA binding activity.

Objective To clone and express the gene encoding ESAT antigen of Mycobacterium tuberculosis (chinese stain). Methods Genome of mycobacterium tuberculosis was extracted. Then, the full length cDNA encoding ESAT protein was amplified by PCR and cloned into prokaryotic expressing vector pGEX5T and sequenced. pGEX5T ESAT was expressed in k802 Ecoli, and the expressed protein was determined by western blot using the sera from ten patients with tuberculosis. Results One specific band of 0.3kb or so was obtained and sequencing result was identical to that reported from Genbank. The expressed protein could be specifically recognized by the sera from tuberculosis patients. Conclusions The full length cDNA of Mycobacterium tuberculosis (Chinese strain) ESAT protein was cloned and expressed successfully, which will be helpful in further studies on diagnosis and treatment of tuberculosis.

OBJECTIVE To detect the expression of the Glucocorticoid receptor-?(GR-?)mRNA in nasal polyp and normal nasal mucosa and investigate the route of nasal drug administration in patients of nasal polyp. METHODS The expression of GR-?mRNA in 101 samples of nasal polyps and 31samples of normal nasal mucosa was examined by using Fluorescent quantitative PCR .RESULTS The level of GR-?mRNA in normal nasal mucosa(135.4? 5.25)?104 copy /?g were significantly higher than that in nasal polyps(23.5?12.1)?104 copy/?g . CONCLUSION Because of the low expression of GR-?mRNA in nasal polyps,the route of nasal drug administration in patients with nasal polyps may be in the part of normal mucosa,not in the mucosa of nasal polyps of the nasal cavity.

Objective To study the changes of proteome expression in brain tissues from rats with severe traumatic brain injury(sTBI). Methods Total protein of brain tissues were obtained at days 3,7 and 14 for two-dimensional gel electrophoresis to screen and identify differential protein spots.Results We screened 17 differential protein spots that were involved in cellular metabolism,stress and inflammatory reaction. Conclusion Some differential proteins involved in sTBI can be found by twodimensional gel electrophoresis.

Objective To locally inject human umbilical cord blood (HUCB) mesenchymal stem cells (MSCs) to rat traumatic brain injury (TBI) model to investigate expression of neural markers and neurological functional improvement. Methods HUCB-MSCs were labeled by bis-benzimide for over 24 hours and stereotactically transplanted into the brain of the rats. All rats were divided into four groups, ie, sham injury group, TBI group, control (TBI + PBS) group and treatment (TBI + MSCs) group, Im-munohistochemical methods and immanofluorescence staining were used to observe the survival, migration and differentiation of the transplanted cells. The neurological functional improvement was evaluated by u-sing the neurological severity score (NSS). Results There existed a large number of MSCs survived in local region of the brain that received transplants, when some MSCs differentiated into neurons or astro-cytes and expressed the neurocyte markers including NSE and GFAP around the grafted site. Treatment group had significantly improved scores compared with sham injury group, TBI group and control group. Conclusions HUCB-MSCs transplantation can potentially improve neurological functional after TBI and may be a good alternative to bone marrow cells for stem cell transplantation or cell therapy.

Objective To explore the correlation between brainstem auditory evoked potential (BAEP) combined with mesencephalic morphology and the prognosis of coma patients with severe craniocerebral injury.Methods Forty-seven coma patients with severe craniocerebral injury were examined with BAEP and moaitored dynamically in early period of coma,and all these patients got head CT scans and the ratio of anteroposterior diameter and transverse diameter of mesencephalon were measured at the same time.Results There was a significantly negative relationship between the first BAEP classification and GOS score at the end of the follow-up (r =-0.755,P =0.000 ).Among all of the patients,there were 27 patients with the ratio of anteroposterior diameter and tansverse diameter of mesencephalon from 0.9 to 1.1,19 patients (70.4％,19/27) with good prognosis;20 patients with the ratio ＞1.1 or ＜ 0.9,6 patients (30.0％,6/20 ) with good prognosis.The rate of good prognosis between the two had significant difference ( x2 =7.521,P =0.006).The sensitivity and specificity were 73.7％(14/19),82.6％ (19/23) at the first BAEP combined with mesencephalic morphology,88.2％ (15/17),100.0％ (21/21) at the second BAEP combined with mesencephalic morphology.Conclusion BAEP combined with mesencephalic morphology can evaluate effectively the prognosis of coma patients with severe craniocerebral injury.

Objective To investigate the association of estrogen receptor-? (ER-?) and vitamin D receptor (VDR) gene polymorphisms with peak bone mass in Shanghai women. Methods The ER-? PvuⅡ and XbaⅠ genotypes and VDR ApaⅠ genotypes were determined by PCR-RFLP in 515 unrelated healthy women aged 19-40 years of Han nationality in Shanghai. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. Results Frequencies of ER-? PvuⅡ genotype PP, Pp and pp were 13.2%, 49.3% and 37.5% respectively. Frequencies of ER-? XbaⅠ genotype XX, Xx and xx were 4.7%, 40.4% and 54.9% respectively. Frequencies of VDR ApaⅠ genotype AA, Aa and aa were 5.8%, 41.9% and 52.3% respectively. Hardy-Weinberg equilibrium was evident for both ER-? and VDR gene polymorphisms. No association was found between ER-? PvuⅡ and XbaⅠ genotypes and BMD of various sites in women. Only a significant association was found between VDR ApaⅠ genotype and BMD at L 1-4(P

In the present study, the clinical features of a patient with autosomal recessive hypophosphatemic rickets 1 caused by dentin matrix protein 1(DMP1)gene mutation and her family members were investigated. DMP1 gene from peripheral blood was sequenced by Sanger sequencing, and the known mutation was verified among her family members and 250 healthy populations. The proband was a 42-year-old female with bone deformity of both lower limbs, bone pain, and short stature. The results of X-rays and laboratory examination were consistent with the hypophosphatemic rickets reported before. A homozygous mutation(c.2T> C)in DMP1 was identified by Sanger sequencing in the proband, her son and daughter were heterozygous for c. 2T> C.