Preparation of Monoclonal Antibody against P65 Protein of Mycoplasma hyopneumoniae

P65 protein, the major immunodominant protein of Mycoplasma hyopneu-moniae （Mhp） exhibiting no cross-reaction with other mycoplasmas, is general y used as a target protein for Mhp detection. In this study, BALB/c mice were immunized with prokaryotical y expressed P65 recombinant protein to prepare monoclonal anti-body. After screening with Mhp whole-cel protein and P65 protein, a specific hy-bridoma cel line, 3G12, was obtained by ELISA. Identification results indicated that the antibody secreted by 3G12 hybridoma cel s could react with P65 protein and Mhp whole-cel protein. According to indirect ELISA assay, 3G12 cel culture super-natant possessed a titer of 1∶12 800 against P65 protein and 1∶3 200 against Mhp whole-cel protein; 3G12 ascites possessed a titer of above 1∶4 000 000 against P65 protein and above 1∶20 000 against Mhp 168 whole-cel protein. After long-term in vitro culture and continuous freezing-thawing, 3G12 cel line could stably secrete antibodies. A monoclonal antibody against P65 protein and Mhp whole-cel protein was successful y obtained in the present study, which provided basis for further in-vestigating the pathogenic mechanism of Mhp and establishing diagnostic methods of Mycoplasmal pneumonia of swine （MPS）.

Mapping of QTLs for Sheath Blight Resistance Using Recombinant Inbred Lines of Rice(Oryza sativa L.)

A recombinant inbred line （RIL） population composed of 157 lines derived from an inter-subspecific hybrid of Daguandao/IR28 by the single seed descent method was used as materials, and the quantitative trait loci （QTLs） coffering the resistance to sheath blight in the 157 RILs and the parents were detected using the toothpick inoculation method. The disease indexes of rice sheath blight in the two parents and 157 RILs were scored and the QTLs responsible for rice sheath blight resistance were detected accordingly by QTL Cartographer software. The results showed that a total of 4 QTLs （qsbl, qsb2, qsb5-1, qsb5-2） conferring sheath blight resistance were detected on chromosomes 1, 2 and 5, and their variance explained ranged from 10.41% to 36.92%. The additive effect of qsb5-1 was negative, indicat- ing that the QTLs derived from donor parent IR 28 could enhance the resistance to sheath blight. However, the additive effects of qsbl, qsb2 and qsb5-2 were positive, indicating that the QTLs derived from donor parent Daguandao weakened the resis- tance to sheath blight.

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185^(c-erbB-2)

The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.

Expression and identification of recombinant soluble single-chain variable fragment of monoclonal antibody MC3

AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.

Indirect ELISA with Recombinant GP5 for Detecting Antibodies to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome is caused by the PRRS virus (PRRSV),which has six structural proteins (GP2,GP3,GP4,GP5,M and N). GP5 and N protein are important targets for serological detection by enzyme-linked immunosorbent assay (ELISA) and other methods. Toward this goal,we developed an indirect ELISA with recombinant GP5 antigens and this method was validated by comparison to the LSI PRRSV-Ab ELISA kit. The results indicated that the optimal concentration of coated recombinant antigen was 0.2 μg/well for a serum dilution of 1:40. The rate of agreement with the LSI PRRSV-Ab kit was 88.7% (266/300). These results support the potential use of recombinant GP5 as an antigen for indirect ELISA to detect PRRSV antibodies in pigs.

CLINICAL EVALUATION OF FOUR RECOMBINANT TREPONEMA PALLIDUM ANTIGEN-BASED RAPID TESTS IN THE DIAGNOSIS OF SYPHILIS

Objective To assess the sensitivity, specificity, and feasibility of 4 recombinant Treponema pallidum antigenbased rapid tests in the diagnosis of syphilis. Methods A total of 970 outpatients were selected from the Sexually Transmitted Diseases Centre of Peking Union Medical College Hospital. Venous blood was collected and serum was extracted. T. paUidum antibodies in whole blood, anticoagulant whole blood, and serum were detected using 4 recombinant T. pallidum antigen-based rapid tests. T. pallidum haemagglutination test （TPHA） was considered as the gold standard for the detection of T. pallidum specific antibodies in serum. The sensitivities and specificities of four methods were analyzed. Results The sensitivities and specificities of Abbott Determine Syphilis TP test, SD-BIOLINE Syphilis 3.0 test, VISITECT-SYPHILIS test, and Syphicheck-WB test for serum specimens were 100% and 98. 9%, 95.7% and 98.0%, 94.6% and 98.2%, 68.1% and 98.9% ; for whole blood were 74. 1% and 99. 5%, 87.9% and 99.4% , 73.2% and 99.7%, 64. 7% and 99.7%. The observed sensitivities of the 4 rapid diagnosis tests were not significantly different with TPHA （ P 〉 0.05 ）. Conclusions The 4 rapid tests show good performance and characteristics in the diagnosis of syphilis. Furthermore, they are more sensitive for serum specimens than whole blood.

Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

AIM： To insert the constructed TGF-β1 epitope gene into the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1 antigenicity in its prokaryotic expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1 vaccine. METHODS： The TGF-β1 encoding epitope gene （the mature TGF-β1 from 78-109 amino acid residues, TGF-β1^32） was amplified by polymerase chain reaction from the recombinant pGEM-7z/ TGF-β1 ^32 vector. The HBcAg gene fragments （encoding HBcAg from 1-71 and 89-144 amino acid residues） were amplified from PYTAI- HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1^32 and HBcAg89-144 into restrictive endonuclease enzyme and ligated with T4 Ugase. The fusion gene fragments HBc.Ag1-71-TGF-β1^32 HBcAg89-144 were recloned to pET28a（＋） and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a （＋）/ CTC was transformed and expressed in E. coli BL21 （DE3） under induction of IPTG. After purification with Ni^＋2 NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope. RESULTS： Enzyme digestion analysis and sequencing showed that TGF-β1 epitope gene was inserted into the el loop of C-terminus of truncated hepatitis B core antigen. SDS-PAGE analysis showed that relative molecular mass （Mr） of the expressed product by pET28a （＋）/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1 polyclonal IgG but not with anti-HBcAg. The purity of protein was about 90 % and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1 antigenicity and could be used as anti-TGF-β1 vaccine. CONCLUSION： A recombinant prokaryotic expression system with high expression efficiency of the target TGF-β1 epitope gene was successfully established. The fusion protein is in the form of self-assembling particles and HBcAg can increase the antigenicity of TGF-β1. The expressed TGF-β1 epitope gene shows good immunogenicity and antigenicity.

The expression and antigenicity identification of recombinant rat TGF-β1 in bacteria

In order to study structure-function details of TGF-beta1, the recombinant mature form of rat TGF-beta1 was expressed in bacteria. Synthesis of the 112 amino-acid carboxyl-terminal part of TGF-beta1 (amino acid 279-390) was controlled by an inducible gene expression system based on bacteriophage T7 RNA polymerase. This system allowed an active and selective synthesis of recombinant TGF-beta1. The molecular weight of expressed TGF-alpha1 monomer determined on SDS-polyacrylamide gel under reducing conditions was about 13 kD. Serial detergent washes combined with a single gel-filtration purification step were sufficient to purify the expression product to homogeneity. Amino-terminal sequencing revealed that the N-terminal of the recombinant protein was identical to the published data. In Western blot analysis the recombinant polypeptide showed excellent antigenicity against polyclonal TGF-beta1 antibody. The mature recombinant rat TGF-beta1 expressed in this study provides a useful tool for future detailed structural and functional studies.

Effects of recombinant human growth hormone on enterocutaneous fistula patients

AIM： To explore the effects of recombinant human growth hormone （rhGH） on intestinal mucosal epithelial cell proliferation and nutritional status in patients with enterocutaneous fistula. METHODS： Eight patients with enterocutaneous fistulas received recombinant human growth hormone （10 ug/d） for 7 d. Image analysis and immunohistochemical techniques were used to analyse the expression of proliferating cell nuclear antigen （PCNA） in intestinal mucosal epithelial cells in biopsy samples from the patients who had undergone an endoscopic biopsy through the fistula at day 0, 4 and 7. Body weights, nitrogen excretion, serum levels of total proteins, albumin, prealbumin, transferrin and fibronectin were measured at day 0, 4 and 7. RESULTS： Significant improvements occurred in the expression of PCNA in the intestinal mucosal epithelial cells at day 4 and 7 compared to day 0 （24.93 ± 3.41%, 30.46 ± 5.24% vs 12.92 ± 4.20%, p 〈 0.01）. These changes were accompanied by the significant improvement of villus height （500.54 ± 53.79 um, 459.03 ± 88.98um vs 210.94 ± 49.16 um, P 〈 0.01）, serum levels of total proteins （70.52 ± 5.13 g/L, 74.89 ± 5.16 g/L vs 63.51 ± 2.47 g/L, P 〈 0.01）, albumin （39.44 ± 1.18 g/L, 42.39 ± 1.68 g/L vs 35.74 ± 1.75 g/L, P 〈 0.01） and fibronectin （236.3 4- 16.5 mg/L, 275.8± 16.9 mg/L vs 172.5 ± 21.4 mg/L, P 〈 0.01） at day 4 and 7, and prealbumin （286.38 ± 65.61 mg/L vs 180.88 ± 48.28 mg/L, P 〈 0.05）, transferrin （2.61 ± 0.12 g/L vs 2.41 ±0.14 g/L, P 〈 0.05） at day 7. Nitrogen excretion was significantly decreased at day 7 （3.40 ± 1.65 g/d vs 7.25 ± 3.92 g/d, P 〈 0.05）. No change was observed in the body weight. CONCLUSION： Recombinant human growth hormone could promote intestinal mucosal epithelial cell proliferation and protein synthesis in patients with enterocutaneous fistula.