Engineered Human Anti-human Antibody Preparation IgE

Objective:This study is prepared to get specific human antibody and humanized mouse monoclonal antibody against IgE C3-C4.Methods:We got human IgE C3-C4gene by PCR, after ligating it with the vector pET-19b, we expressed it in E. coli strain BL21(DE3) star pLysS. We established a human IgG genes library and screened it with recombinant IgE C3-C4by using cloning blotting assay and then evaluated the screened antibody by enzyme linked immunosorbent assay (ELISA), Western bloting, Biacore and histamine release inhibit test, and then engineered the antibody with chain reshuffling and site-directed mutagenesis to get more efficient antibodies. At the same time, we immuned BALB/c mouses with recombinant IgE C3-C4, and then fused mouses’ spleen cells with mouses’myeloma cells to obtain hybridoma cells. We got the monoclonal antibodies through mouses’ascites and then humanized the antibody.Results:We have successfully got the recombinant IgE C3-C4and established a human IgG genes library with a titer of9×107. And we succeeded to get a specific antibody to IgE C3-C4by cloning blotting assay which had good reactivity evaluated by in vitro tests. We got one chain reshuffling antibody and three site-directed mutagenesis antibodies and they both had good reactivities. We got5lines of hybridoma with good reactivity and humanized2of them.Conclusion:We got a human antibody and5lines of mouse monoclonal antibodies to IgE C3-C4, they all had good reactivities. We could do more in vitro and in vivo researchs to find out its valuable usage. Objective:Amebiasis caused by Entamoeba histolytica is one of the most problematic parasitic diseases in developing and developed countries. We conducted this survey to get an overview of seroprevalence of Entamoeba histolytica infection in China.Methods:We prepared crude antigen from Entamoeba histolytica strain HK9trophozoites and expressed recombinant Igl-C (C-fragment of Entamoeba histolytica Gal/GalNAc inhibitable lectin intermediate subunit) in E.coli strain BL21(DE3)pLysS. Then1312serum samples were tested by ELISA using crude antigen and Igl-C, and a logistic regression model was established.Results:A total of1312(M:678, F:634) serum samples from seven provinces were tested by ELISA and the positive rates regarding to Igl-C test were as follows:1.06%(2/188) for Beijing,3.85%(5/130) for Shanghai,7.04%(10/142) for Sichuan,3.17%(6/189) for Guangxi,14.39%(41/285) for Guizhou,0.53%(1/190) for Qinghai and9.04%(17/188) for Sinkiang. The logistic regression model was as follows:Conclusion:This is the first report on seroprevalence of E. histolytica infection in China and the result is in good accordance with the first national parasitic diseases investigation in1988-1992. The logistic regression model can provide prior probability before any clinical experiment, which is helpful to further schedule of experiment. Objective:Identification of astigmatid mites based on their morphological characters is difficult because of the similarity of their organs, especially in immature mites. Our objective is to find an easy way to identify astigmatid mites.Methods:We got the genomic DNA of mites after morphologically identification and then amplified the ribosomal second internal transcribed spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) regions by PCR. After aligning the sequences by using ClustalX, we calculated intra-and interspecific genetic distances by PHYLIP and got phylogenetic trees by using neighbor-joining and maximum parsimony methods.Results:The ITS2and COI regions of six species of astigmatid mites (Aleuroglyphus ovatus, Blomia tropicalis, Dermatophagoides farina, Dermatophagoides pteronyssinus, Euroglyphus maynei, Tyrophagus putrescentiae,) were obtained by polymerase chain reaction and sequenced. The lengths of the ITS2sequences varied from316to488bp, while the COI regions were377or378bp long. Considering the ITS2genes, the intraspecific genetic distance was in the range of0.00-0.077844, whereas the interspecific genetic distance was0.202426-0.912959. The values were0.000-0.029748and0.138403-0.279304for intra-and interspecific genetic distances when COI genes were used. The phylogenetic trees inferred from the ITS2and the COI regions, by using maximum parsimony and neighbor-joining methods, were identical to those based on their morphological classification.Conclusion:The ITS2and COI regions can be applied as barcodes to identify different species of astigmatid mites.