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Cloning and eukaryotic expressing of GPI-B7-1 in CHO

Luo Chao-quan

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Abstract

AIM: To construct human GPI-B7-1 fusion protein and investigate the therapeutic potentials in the treatment of tumors. METHODS: The chimeric GPI anchored-B7-1 gene was obtained by overlap PCR and inserted into expressing vector pcDNA3.1, named pc3.1/GPI-B7-1. pc3.1/GPI-B7-1was transfected into CHO cells by lipofectamine ~2 000 reagent. The CHO cells, expressing GPI-B7-1 on membranes, were obtained after selecting by G418. That was confirmed by flow cytometry, SDS-PAGE and Western blot. RESULTS: Recombinant vector pc3.1/GPI-B7-1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected CHO cell membrane selected by G418 was confirmed to be GPI-anchored protein by flow cytometry, and GPI-B7-1 approximately 60 kD was conformed by SDS-PAGE and Western blot. CONCLUSION: A large amount of GPI-B7-1 fusion protein was obtained and will be further studied in the treatment of tumors.2? [

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AIM: To construct human GPI-B7-1 fusion protein and investigate the therapeutic potentials in the treatment of tumors. METHODS: The chimeric GPI anchored-B7-1 gene was obtained by overlap PCR and inserted into expressing vector pcDNA3.1, named pc3.1/GPI-B7-1. pc3.1/GPI-B7-1was transfected into CHO cells by lipofectamine ~2 000 reagent. The CHO cells, expressing GPI-B7-1 on membranes, were obtained after selecting by G418. That was confirmed by flow cytometry, SDS-PAGE and Western blot. RESULTS: Recombinant vector pc3.1/GPI-B7-1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected CHO cell membrane selected by G418 was confirmed to be GPI-anchored protein by flow cytometry, and GPI-B7-1 approximately 60 kD was conformed by SDS-PAGE and Western blot. CONCLUSION: A large amount of GPI-B7-1 fusion protein was obtained and will be further studied in the treatment of tumors.2? [

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Available abstract

AIM: To construct human GPI-B7-1 fusion protein and investigate the therapeutic potentials in the treatment of tumors. METHODS: The chimeric GPI anchored-B7-1 gene was obtained by overlap PCR and inserted into expressing vector pcDNA3.1, named pc3.1/GPI-B7-1. pc3.1/GPI-B7-1was transfected into CHO cells by lipofectamine ~2 000 reagent. The CHO cells, expressing GPI-B7-1 on membranes, were obtained after selecting by G418. That was confirmed by flow cytometry, SDS-PAGE and Western blot. RESULTS: Recombinant vector pc3.1/GPI-B7-1 was successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected CHO cell membrane selected by G418 was confirmed to be GPI-anchored protein by flow cytometry, and GPI-B7-1 approximately 60 kD was conformed by SDS-PAGE and Western blot. CONCLUSION: A large amount of GPI-B7-1 fusion protein was obtained and will be further studied in the treatment of tumors.2? [

Key concepts: Lipofectamine, Transfection, Molecular biology, Western blot, Flow cytometry, Recombinant DNA, Chinese hamster ovary cell, Fusion protein

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