Results

1. Construction of recombinant plasmid of HCV Core protein

1.1 Codon optimization of HCV core protein

tu1,2

Fig.1 Part sequence of original (left) or optimized (right) HCV core protein at website http://gcua.schoedl.de/sequential_v2.html

optimized sequence of HCV core gene was showed as follows:

GGTACCATGAGTACCAATCCGAAACCGCAGCGCAAAACCAAACGTAATACCAATCGTCGTCCGGAAGATGTTAAATTTCCGGGCGGCGGTCAGATTGTGGG

CGGCGTTTATCTGCTGCCGCGTCGTGGCCCGCGTCTGGGTGTTCGTACCACCCGTAAAACCAGTGAACGCAGTCAGCCGCGCGGCCGCCGTCAACCTATTC

CGAAAGATCGTCGCAGTACCGGCAAAGCCTGGGGCAAACCGGGCCGTCCGTGGCCTCTGTATGGTAATGAAGGTCTGGGCTGGGCCGGTTGGCTGCTGAGC

CCTCGTGGTAGTCGTCCGAGTTGGGGCCCGACCGATCCGCGTCATCGCAGTCGTAATGTGGGTAAAGTGATTGATACCCTGACCTGTGGCTTTGCAGATCT

GATGGGCTATATTCCGGTGGTTGGCGCACCGCTGAGCGGTGCAGCACGCGCAGTTGCACATGGCGTTCGTGTTCTGGAAGATGGTGTTAATTATGCCACCG

GCAATCTGCCGGGCTTTCCGTTTAGTATTTTTCTGCTGGCCCTGCTGAGCTGTATTACCGTGCCGGTGAGCGCCCTGCAG

1.2 PCR and confirmation of HCV C gene

For expression efficiently, the optimized and truncated HCV C genes, O173 (173AA) and O120 (120AA), were amplified using PCR method. The primers and PCR results are showed as below:

tu3,4

Fig.2 Recycled enzyme-digested PCR products.

1.3 O173 and O120 genes were cloned into pColdII vector

tu5

Fig.3 Before ligation of PCR products and pColdII, they were ldigested by restriction enzymes.

1.4 Identification of pColdII-O120 and pColdII-O173 recombinant plasmid

After ligation reactions, the products were transformed into the competent cell DH5α. We used colony PCR method to select the positive clones, and then send them to the company for sequencing. Colony PCR results were showed as below:

tu6.1

Fig.4 Colony PCR products to identify the recombinant plasmids pColdII-O120 and pColdII-O173

tu6