Experimental study on bacteriostatic effect of recombinant human lactoferrin on Helicobacter pylori / 重庆医学

Objective To evaluate the bacteriostatic effect of recombinant human lactoferrin(rhLF) on Helicobacter(H .) py‐lori and its influence on CagA ,Ure and gastric mucosal IL‐8 .Methods The minimum inhibitory concentration(MIC)and the influ‐ence of different drug concentrations on the proliferation of H .pylori were detected .The effects of rhLF on the mRNA and protein expressions of CagA and Ure in H .pylori were detected by RT‐PCR and Western blot ,respectively .The animal study :Balb/c mice were adopted and assigned randomly into four groups ,including the standard triple+rhLF(group A) ,rhLF(group B) ,standard tri‐ple(group C) and normal saline(group D) .The histopathological HE staining was used to observe the gastric inflammation and ELISA was used to detect the IL‐8 level of gastric tissue in each group .Results MIC was 0 .5 mg/mL ,moreover rhLF inhibited the bacterial growth and proliferation with a concentration‐dependent manner .rhLF could reduce the expression of H .pylori major viru‐lence factor CagA ,mRNA and protein of Ure .Comparing the group A with the group B ,C and D ,the gastric mucosal inflammation score and the IL‐8 levels of gastric tissue homogenates had statistically significant differences(P<0 .05) .Conclusion rhLF inhibits the growth and proliferation of H .pylori ,moreover inhibit the expression of major virulence factor CagA in H .pylori ,mRNA and protein of Ure in different degrees ,weakens its pathogenicity ,meanwhile reduces the IL‐8 level in mice gastric mucosa ,and allevi‐ates H .pylori related gastric mucosal inflammatory response .

Deletion of marker gene in transgenic goat by Cre/LoxP system / 生物工程学报

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.

Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity / 生物工程学报

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

Generation of fully human anti-vascular endothelial growth factor 165 monoclonal antibody with antitumor efficacy from transgenic five-feature mice / 中国药科大学学报

Aim: To prepare a fully human anti-VEGF_(165)(vascular endothelial growth factor 165) monoclonal antibody with antitumor activity from five-feature mice which express human immunoglobin loci. Methods: A routine method for the generation of monoclonal antibodies( mAbs) against the human VEGF_(165) was developed. The immunizing effect between five-feature mice and BALB/c was observed and the mAb was purified through MBP IgM affinity chromatography. The effect of mAbs on antitumor was tested ire vitro by T24 cell line. Results: Four hybri-domata secreting mAbs steadily were isolated successfully, and the serum titer of mAb in BALB/c mice was almost 10 times higher than that in five-feature mice. The indirect ELISA method for mAb titer determination was also established. The anti-VEGF_(165) mAb was purified to homogeneity by precipitation with ammonium sulfate followed by the affinity chromatography on MBP IgM purification column. Moreover, both purified human IgM V_2, V_(75) and mouse ascites were characterized by SDS-PAGE and Western blotting. Proliferation of T24 cell line was considerably inhibited by V_2 and V_(75). Conclusion: Five-feature mice could be used to produce fully human monoclonal antibody. The fully human anti-VEGF mAb is potential in the cancer treatment.

Prediction,synthesis and characteristics of B-cell epitopes of CSFV envelope glycoprotein E2 / 中国免疫学杂志

Objective:Study on characteristics of two synthesizd peptides based on CSFV E2 protein. Methods:B cell epitopes of CSFV E2 antigen were predicted using accessibility and flexibility schemes, associated with antigenicity , secondary structure and multiple sites prediction. Two antigen peptides (Pep1 and Pep2) have been designed and synthesized and their reactivety were detected with 8 McAbs and antiserum against mE2 protein, then the peptides were conjugated with BSA and immunized rabbits respectively. Results:Both Pep1 and Pep2 could react with antiserum and McAb A11, Pep2 could interact with McAbD5 and McAbD8. Only Pep1 BSA conjugate can stimulate high level and specific antibodies.Conclusion: The peptide1 has good antigenicity.

Cloning, Sequencing and Expression of Trichinella spiralis p49 Gene / 中国寄生虫学与寄生虫病杂志

Objective To conduct cloning, sequencing and expression of Trichinella spiralis ES antigen p49 gene. Methods RT-PCR was used to amplify the specific gene fragment from the total RNA of Trichinella spirais larvae. The PCR product was ligated to the T-vector and the recombinant plasmid was verified by sequencing. T-p49 and pGE-4T-3 were treated by both BamHI and XhoI. The ligation reaction was catalyzed by T4 DNA ligase. Results The p49 gene was cloned by using RT-PCR. Sequence analysis showed that the p49 gene obtained was consistent to the p49 sequence reported in the database. The expressed protein was shown as a new band at SDS-PAGE. BLAST analysis demonstrated that this p49 gene was 99% identical to the p49 gene reported and to the 43 kDa secreted glycoprotein gene in the database. Conclusion p49 gene from Trichinella spiralis larvae was cloned, sequenced and expressed.