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Fan J.-H.,Huangpu District Center Hospital | Cheng G.-X.,Shanghai Transgenic Research Center
Chinese Journal of Cancer Prevention and Treatment | Year: 2012

OBJECTIVE: To prepare of EGFRv III Monoclonal Antibody, and it is expected to be good to the tumor's target treatment. METHODS: The peptide of EGFRv III was synthesized, and conjugated with KLH. Balb/c mouse was immuned, hybridoma cell was used to generate ascites, antibody from ascites was purified and the speciality of the monoclonal antibody was determined. RESULTS: The corresponding 14-amino acid peptide of the gene absence fusing area of EGFRv III, and conjugatd with KLH. Immune Balb/c mouse using this conjugation as antigen with a certain immune protocal, when high titer antiserum (1:128 000) was obtained, fuse the spleen cell of immuned mouse with SP2/0 cell, select by ELISA and subclone to obtain anti EGFRv III stabilized hybridoma cell lines. 5 mouse hybridoma lines obtained and ascites produced. The type of Monoclonal Antibody is IgG2a, Determine ascites antibody titer and it's affinity with antigen. The titer was 1:128000, and the highest affinity of those was 9.8×10 -9 mol/L. The speciality of the monoclonal antibody was determined by the expressed natural EGFR's binding domain in Pichia yeast. The results showed that the antibody could not bind with the expressed peptide. It indicated that the monoclonal antibody prepared by ourselves was special to EGFRv III. CONCLUSION: The succeeding preparation of EGFRv III monoclonal antibody by this experiment gives a stability base to the tumor's target treatment.

Yao L.,Chongqing Medical University | Wang P.,Fudan University | Liu J.,Chongqing Medical University | Chen J.,Shanghai Transgenic Research Center | And 2 more authors.
International Journal of Clinical and Experimental Medicine | Year: 2014

Poor development of the interspecies somatic cell nuclear transfer (iSCNT) embryos was due to nuclear-mitochondrial incompatibility. In humans, it has been known that ooplast transfer (OT) could support normal fertilization, the development of embryos and prevents the transmission of mtDNA disease. To investigate whether OT could support development of the iSCNT embryos, the ooplast of Triploid Pronucleus (3PN) zygote which would be discarded in IVF lab was transferred into the enucleated goat oocytes to construct humanized iSCNT embryos in our study. The results showed the 3PN-OT could significantly improve the early development of humanized iSCNT embryos. The percentage of blastocyst development of OT group was also higher than that of the control group. Interestingly, the morphology of some OT-iSCNT blastocysts was similar to normal human blastocysts in vitro fertilization, while the morphology of iSCNT blastocysts from control group was similar to goat blastocysts. Importantly, the pluripotent marker Oct4 of the OT-iSCNT blastocyst was expressed stronger than that of the control group. These results suggested that 3PN-OT could improve the developmental potency of human iSCNT embryos and would facilitate establishing ESCs from iSCNT blastocysts. © 2014 E-Century Publishing Corporation. All rights reserved.

Bao Z.,Nanjing Agricultural University | Gao X.,Nanjing Agricultural University | Zhang Q.,Nanjing Agricultural University | Lin J.,Nanjing Agricultural University | And 5 more authors.
PLoS ONE | Year: 2015

The development of genetically engineered animals has brought with it increasing concerns about biosafety issues. We therefore evaluated the risks of growth hormone from transgenic goats, including the probability of horizontal gene transfer and the impact on the microbial community of the goats' gastrointestinal tracts, feces and the surrounding soil. The results showed that neither the GH nor the neoR gene could be detected in the samples. Moreover, there was no significant change in the microbial community of the gastrointestinal tracts, feces and soil, as tested with PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing. Finally, phylogenetic analysis showed that the intestinal content, feces and soil samples all contained the same dominant group of bacteria. These results demonstrated that expression of goat growth hormone in the mammary of GH transgenic goat does not influence the microflora of the intestine, feces and surrounding soil. © 2015 Bao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Guan X.-H.,Shanghai JiaoTong University | Feng Y.,Shanghai JiaoTong University | Lu X.-W.,Shanghai JiaoTong University | Chen J.-Q.,Shanghai Transgenic Research Center | And 2 more authors.
Journal of Reproduction and Contraception | Year: 2013

Objective: To analyze the blastocyst formation and chromosome statuses of reconstructed embryos derived from human-goat interspecies somatic cell nuclear transfer (iSCNT), exploring the development retardant factors. Methods: Human specific point probes cep2, cep6, tel2 and 13q14.2, 21q22.13 combining fluorescence in-situ hybridization (FISH) technology were used to test trophectoderm cells of blastocyst and blastomeres of development arrest nuclear transfer (NT) embryos. Results: A total of 209 reconstructed embryos were recovered, and the rate of blastocyst formation was 3.8% (8/209). FISH signals showed that chromosomal abnormalities were present in 2 blastocysts (2/8) and 146 development arrest embryos (146/201). Conclusions: The rate of blastocyst formation is low, and reconstituted embryos of development arrest showed extensive chromosome abnormalities, suggesting that a chromosomal mechanism may underlie their developmental arrest. © 2013 The Editorial Board of Journal of Reproduction and Contraception.

Liu H.,Tongji University | Liu H.,Shanghai Transgenic Research Center | Liu S.,Shanghai Transgenic Research Center | Wu Y.,Shanghai Transgenic Research Center | And 6 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2010

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. © 2010 CJB, All rights reserved.

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