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| Construction and Biological Assay of Integrated Interferon Mutant IIFN/165S |
| TIAN Shuo1, YAO Wen-bin2, XU Chen3 |
1. Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, National Laboratory of Medical Molecular Biology, Beijing 100005, China;
2. School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, China;
3. Beijing Tri-prime Genetic Engineering Co.Ltd., Beijing 102600, China |
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Abstract Objective: Construct IIFN/165S through site-directed mutagenesis in order to obtain a new type of molecule with higher potency. Methods: CGT was substituted for AGT at position 165 of integrated interferon through PCR site-directed mutagenesis in vitro. The Amplified fragment was constructed in pET23b expression vector, and transformed into E. coli BL21 (DE3) pLysS. The recombinant protein was purified and the purified protein was analyzed by SDS-PAGE, Western blot and MALDI-TOF-MS. The anti-virus was determined by WISH-VSV system. The apoptosis rate was detected by flow cytometry. Result: IIFN/165S was expressed as inclusion bodies with the yield of more than 30% of total bacterial protein. The purity of IIFN/165S was more than 95% with IIFN immunogenicity, and the molecular weights of IIFN/165S was 18172. The biological activity was (7.63±0.22)×108×106 IU/mg. IIFN/165S induced Daudi cells apoptosis in a dose-dependent manner. Conclusion: The construction, expression and purification technology of IIFN/165S had been established.
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Received: 11 December 2012
Published: 25 July 2013
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