TNF? mRNA in human bronchoalveolar lavage(BAL)cells was quantitated by a olymerase chain reaction(PCR)using a cRNA internal standard,cRNA molecules were in vitro transcribed from pAW108 plasmid in which the sequences of upstream primer and omolementary sequences of downstream primer of TNF? were inserted.The cRNA and mRNA extracted from BAL cells were mixed and reverse-transcribed into cDNA.The resultant cDNA mixture was 1:3 diluted and amplified by PCR using TNF? specific primers.~(32)p-labeled upstream primers were included in the PCR reaction,cDNA fragments amplified was run on a 3% agarose gel.The radioactivity of positive bands was determined in a scintillation ounter.After plotting variable template concentrations of the internal standard pAW108 cRNA and the number of BAL cells against the radioactivity of their PCR products,the levels of TNF? mRNA in BAL cells were quantitated by comparision to those of cRNA internal standard.

Objective:To clone and express mouse fibroblast growth factor eight(FGF 8) and investigate the function of it Methods:According to the published sequence of mouse fibroblast growth factor 8, a pair of special primers was designed Then the full length cDNA of FGF 8b (a predominant isoform of FGF 8) was isolated from the total RNA of the mouse embryo by means of reverse transcription PCR and subcloned into the yeast expression vector of pYEX4T 1 in correct direction It was identified with sequencing The recombinant plasmid was transformed into yeasts The fusion protein expressed was verified by using Western blot The activity of the protein was examined by MTT and 3H TdR incorporation Results:The cDNA fragment was about 600 bps, and proved to be "b" isoform of FGF 8 exactly This fusion protein, with molecular weight about 55 kD, was highly expressed in yeast system The data of MTT and 3H TdR incorporation in NIH3T3 cells were increased obviously by the protein, and decreased by the antagonist Conclusion:Fibroblast growth factor eight was cloned and expressed.The protein was found to be capable of stimulating the proliferation of NIH3T3 cells Moreover, the recombinant protein of the extracellular fragment of FGFR1 can antagonize the response of NIH3T3 to the recombinant protein

Abstract Objective:To construct bmcella melitensis Br.melitensis genomic DNA expression library and study its immune effect.Meth-ods: Hind Ⅲ digested Br.melitensis genomic DNA fragments were cloned into pSV-?-Gal plasmds.The recombinant plasmids were transformedinto JM109 host bacteria. After large-scale isolation, the recombinant plasmids were inoculated into the quadriceps muscle of mice. In the 9thday after third boost immunization .agglutination antibodies against Br. melitensis and lymphocyte mitogenic ability of mice were determined. Re-sults : Expression library nucleic acid vaccine can induce the production of agglutination antibodies against Br. melitensis and stimulate the lym-phocyte porliferation primed by ConA. Conclusion:The genomic DNA expression library may provide a new vaccine against Br. melitensis infec-tion.

Objective To investigate the value of serum thyroglobulin (Tg) and antithyroglobulin antibody (TgAb) in differentiated thyroid carcinoma complicated with Hashimoto's thyroiditis after thyroid ablation.Methods Serum Tg and TgAb levels and the status of illness in 154 differentiated thyroid carcinoma patients with coexistent Hashimoto's thyroiditis and confirmed pathology after surgery followed by remnant ablation were performed during three years follow up.Tg and TgAb levels were assessed by chemiluminescent immunoassay assay.The cases were divided into three groups (according to the level of Tg):Tg ≤ 1 μg/L group,1 μg/L＜Tg ≤ 10 μg/L group and 10 μg/L＜Tg≤ 100 μg/L group.TgAb＞40 kIU/L was considered as positive,Cox's proportional hazard model was used to analyse prognostic value in different levels of Tg and TgAb for disease-free survival and recurrence.Results Compared with 1 μg/L＜Tg≤ 10 μg/L group and 10 μg/L＜Tg≤ 100 μg/L group,the relative risk in reflecting cancer recurrence (TgAb＞40 kIU/L) in Tg ≤ 1 μg/L group was 27.000 (95 ％ CI 6.727-108.374).The value of TgAb＞40 kIU/L in Tg≤ 1 μg/L group was greatly increased and highly correlated with metastasis.However,In the condition of Tg＞ 1 μg/L,the disease will be based on the level of TgAb.Conclusion The value of TgAb＞40 kIU/L in Tg ≤ 1 μg/L group seems to be the optimal cutoff value correlated with recurrence and metastasis of differentiated thyroid carcinoma.

Objective To produce prokaryotic recombinant protein glyceraldehyde-3-phosphate dehydrogenase of Clonorchis sinensis (CsGAPDH), analyze its enzyme activity and immunological function. Methods The recombinant CsGAPDH was purified according to the protocol of GST?Bind~TM kit and was digested with thrombin proteinase and eluted with wash buffer. The BALB/c mice were inoculated with the purified protein. The antisera collected from the mice were used to detect the titres of IgG antibodies by ELISA, and Western blotting was used to identify the specificity of the antisera with the purified CsGAPDH. S-P immunohistochemistry method was used to confirm the expression and distribution of CsGAPDH in adult Clonorchis sinensis with the polyclonal antibodies from immunized BALB/c mice. The CsGAPDH catalytic activity was evaluated employing the conventional substrate glyceraldehydes-3-phosphate (3-GAP). Results SDS-PAGE showed a single purified protein band. Gel scanning analysis revealed that the protein purity of CsGAPDH was 90%. ELISA analysis showed an increased IgG value. S-P immunohistochemistry analysis demonstrated that the recombinant plasmid pGEX-4T-1-GAPDH expressed and distributed in muscle cell membrane of immune mice. Western blotting result suggested that CsGAPDH protein contained essential epitopes with high antigenic activities. This protein CsGAPDH could catalyzed 3-GAP with enzymatic active unit of 2 872 U min~-1ml~-1. Conclusion The recombinant protein CsGAPDH shows a proper enzymatic activity and immunogenicity.

Objective: To express recombinant human soluble fibroblast growth factor receptor 1 ( sFGFR1) and study its antagonistic activity on FGF. Methods: Human sFGFR1 cDNA, isolated from human lung fibroblast cells with RT-PCR was confirmed by DNA sequencing and cloned into pYEX4T-1 yeast expression vector. The recombinant sFGFR1 was expressed in DY150 yeast cells and the product was identified by SDS-PAGE and Western blot. The activity of recombinant sFGFR1 was detected in N1H3T3 proliferation inhibition assay. Results: GSF-sFGFR1 fusion protein was expressed in yeast cells and was observed as a band of 60 Id) on a SDS- PAGE gel and by Western blot. The recombinant fusion protein was also found to be able to suppress FGF-induced proliferation of NIH3Th cells. Conclusion: Recombinant human GST-sFGFR1 fusion protein was expressed in yeast efficiently and showed natural biological activities.

Objective To find the changes of human fibroblast growth factor receptor 1 genone during development.Method Southern blot analysis of genomic DNA isolated from adult and fetal tissues. Result Adult FGFR1 gene structure is different from its embryonic counterpart. Conclusion The disserence might lead to changes of FGFR1 expression as wel as functions of the cells.

Objective: To express recombinant human angiostatin for further application in clinic. Methods: The complete encoding eDNA of human angiostatin was isolated from human embryo liver with RT-PCR and expressed in secretory Pichia expression system. Recombinant human angiostatin was purified with heparin sepharose chromatography and its activity was determined in chick embryo chorioallantoic membrane (CAM) and wound healing assays. Results: Expressed in large quantity (yield=5 mg/L) and purified with heparin sepharose, recombinant angiostatin was showed to have a molecular weight of 43 kD in SDS-PAGE and potently inhibit angiogenesis and wound healing. Conclusion: Recombinant human angiostatin was expressed efficiently in a biologically active form.

Using human acidic fibroblast growth factor Specific primers we tested the technique of screening cDNA library by a DNA polymerase chain reaction.With the method,we have suc-cessfully isolated encoding region cDNA of human interleukin 8 from both human dermal fibrob-last and PMA stimulated U937 cell cDNA libraries.

Objective:To study the clinical application potential of aFGF and try to produce aFGF as gene engineering medicine.Methods:The complete encoding cDNA of human aFGF was isolated from human lung fibroblast with RT PCR.Recombinant human aFGF was expressed in secretory pichia expression system and purified with heparin sepharose chromatography.The activity of aFGF was detected in NIH3T3 proliferation assay,chick embryo chorioallantoic membrane assay(CAM) and wound healing assay.Results:The recombinant aFGF was expressed in large quantity(yield=12 mg/L) and was capable to promote proliferation of NIH3T3,angiogenesis and wound healing.Furthermore,those activities of aFGF could be agonized by recombinant FGFR extracellular domain.Conclusion:Recombinant human aFGF was expressed efficiently and possessed natural biological activities.