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Chinese Journal of Biotechnology ; (12): 1002-1014, 2012.
Article in Chinese | WPRIM | ID: wpr-342421


To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2 positive cancer cells, indicating a potential candidate method for clinical HER2 positive specimens detection.

Animals , Breast Neoplasms , Diagnosis , Pathology , Female , Genetic Vectors , Genetics , Green Fluorescent Proteins , Genetics , Humans , MCF-7 Cells , Receptor, ErbB-2 , Recombinant Fusion Proteins , Genetics , Sf9 Cells , Single-Chain Antibodies , Genetics
Article in Chinese | WPRIM | ID: wpr-415746


Objective To investigate the genotypes of host killing genes and their single nucleotide polymorphisms (SNPs). Methods Three hundred and twenty strains of Escherichia coli that collected from the First Affiliated Hospital of Wenzhou Medical College were analyzed. The first sample ( E1 ) contains 160 strains isolated during the years from 2002 to 2003. The second sample (E2) contains 160 strains covering the years from 2008 to 2009. The plasmids of Escherichia coli were extracted by alkaline lysis method. Solexa/Illumina sequencing technology was used to sequence plasmids metagenome. Solexa Genome Analysis System and Soap programs were used to analyze gene distribution, SNPs and lineage-specific mutations. Results 11 077 768 reads were generated and 0. 045% of them can map to the reference sequences from El sample. Whereas 9 377 792 reads were generated and 0. 053% of which mapped to the reference from E2 sample. There are nine host killing genes identified in the two samples, of which hok gene is the most prevalent. A total of 29 SNP sites dispersed in five genes of the two samples. Approximately 33% of them were non-synonymous mutations. One position of A and G is the most prevalent polymorphism. Conclusion The known nine genotypes of host killing genes were all identified in plasmids of Escherichia coli in Wenzhou. hok gene showed the highest frequency. There were SNPs in five genotypes.