HPV18 type l2ne7e6 fusion protein gene, expression vector, method, bacterial strain and application
A 18L2NE7E6, L2NE7E6 technology, applied in the field of human papillomavirus type 18 L2NE7E6 fusion protein, can solve the problem of less research
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Embodiment 1
[0057] Example 1: Designing the optimized codon gene sequence of human papillomavirus type 18 L2NE7E6 fusion protein expressed in E. coli
[0058] First, the HPV18 sequence isolated and sequenced from cervical cancer patients was aligned with the sequence in Genebank, which was consistent with AY262282, and then the nucleotide sequence of the minor capsid protein L2N protein was fused with the early protein E7 and E6 nucleotide sequences. And remove the start codon of E7 and E6 proteins and the stop codon of E7, and consider that E7 and E6 proteins are HPV18 oncoproteins. For the safety of the vaccine, the amino acids related to the oncogenic site are taken into account. Mutation was performed by mutating Cys at position 27 and Glu at position 29 of E7 protein to Gly, and the amino acid at position 65 of E6 was mutated from Cys to Gly, and the amino acid sequence encoding the HPV18L2NE7E6 fusion protein was designed to obtain the specific SEQ ID NO. : the fusion protein shown ...
Embodiment 2
[0060] Example 2: Synthesis of codon-optimized gene sequences and construction of cloning plasmids containing 18L2NE7E6 target gene fragments
[0061]The plasmid pGH-18-33 was synthesized by Beijing Qingke Biotechnology Co., Ltd., which contains the nucleotide gene fragment of HPV18L2 protein from positions 1 to 600 designed according to the dominant codon of Escherichia coli, and the L2N protein was first increased by PCR method. Gene fragment, the upstream primer P1: L2F contains 6 histidines and Nde I restriction site, and the downstream primer P2: L2R contains BamH I restriction site, the primers are provided by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Synthesized by the company (the specific sequences of primers are shown in Table 1). Then the amplified PCR fragment was inserted into the cloning vector pMD18-T to construct pMD18-HPV18L2N. The sequence of the HPV18L2N gene was confirmed to be consistent with the designed sequence by sequencing. The spec...
Embodiment 3
[0070] Example 3: Construction of prokaryotic expression recombinant plasmid pET9a-HPV18L2NE7E6
[0071] This example is to construct a prokaryotic expression recombinant plasmid pET9a-HPV18L2NE7E6, which is described in detail as follows.
[0072] The pMD18T-HPV18L2NE7E6 cloning plasmid constructed in Example 2 was digested with enzymes to obtain
[0073] The HPV18L2NE7E6 gene fragment, the specific operation of the enzyme digestion and digestion is as follows: first, digest with Nde I enzyme at 37°C for 2 hours, then recover with agarose gel recovery kit, and then digest with BamH I enzyme at 37°C for 2 hours, and finally use The HPV18L2NE7E6 fragment was recovered by agarose gel recovery kit.
[0074] Then the obtained HPV18L2NE7E6 fragment was inserted into the Nde I and BamH I sites of the E. coli expression plasmid pET9a, identified and sequenced by Nde I and BamH I digestion, and screened to obtain the correct inserted prokaryotic expression recombinant plasmid pET9a-H...
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