Carcinogenesis, Teratogenesis & Mutagenesis

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Identification of the alternatively spliced variant v7 of Zwint-1 in HeLa cells

SU Jing-hua,LI Rui,LIU Jun,ZHOU Yan-lin,CHEN Xiao-xuan,DAI Jian-ping,WANG Ge-fei,LI Kang-sheng   

  1. Department of Microbiology and Immunology, The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou 515041, Guangdong, China
  • Received:2014-01-03 Revised:2014-02-21 Online:2014-03-30 Published:2014-03-30
  • Contact: 李 蕊,E-mail:neery007@163.com
  • About author:苏景华(1989- ),男,福建龙岩人,硕士研究生,研究方向:神经免疫和抗感染免疫。E-mail:breezeflsz@gmail.com
  • Supported by:

    国家自然科学基金资助项目(31300761)

Abstract:

OBJECTIVE: To identify whether the mRNA and protein expression of Zwint-1 v7 existed in human cancer cell line. METHODS:To identify the mRNA with the deleted fragment in HeLa cells,we designed primers on the each side of the deleted fragment for PCR and sequencing. Therefore,we constructed prokaryotic expression vector pGEX-KG-GST-Z7,the inserted fragment Z7 was chosen basen on the different sequences between Zwint-1 and Zwint-1 v7. This vector was transformed into BL21 strain for the expression of fusion protein GST-Z7. After ultrasonication and purified from the SDS-PAGE gels,GST-Z7 was used as antigens and emulsified with adjuvants to immunize C57BL/6 female mice for the preparation of polyclonal antibody Anti-Z7. The serum titer was validated by Dol blot test. The Zwint-1 v7 protein in the total proteins of HeLa cells was evaluated by Western blot with Anti-Z7. RESULTS:The PCR and sequencing results showed mRNA with 37 bp deletions in HeLa cells. SDS-PAGE confirmed that GST-Z7 was correctly expressed in BL21 in the inclusion body. 12 ng GST-Z7 could be detected by the 5 000 times diluted Anti-Z7. Zwint-1 v7 from HeLa cells could be detected by the 200 times diluted Anti-Z7. CONCLUSION:We confirmed that Zwint-1 v7 protein existed in HeLa cells at the levels of translations of nucleic acid and protein structure.

Key words: HeLa cells, Zwint-1 v7 protein, prokaryotic expression, antibody production