HPV58 L1 gene, vector, strain and expression method
A gene and strain technology, applied in chemical instruments and methods, microbial-based methods, botanical equipment and methods, etc., can solve the problems of low expression level and no expression, achieve high expression level, high expression level, and benefit large-scale The effect of large-scale industrial production
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Embodiment 1
[0040] Example 1: HPV58 L1 codon optimization design
[0041] According to the wild-type HPV58 L1 amino acid sequence (GenBank: D90400.1, SEQ ID NO: 2, figure 1 ) to obtain the wild-type 58L1 nucleotide sequence. The wild-type HPV 58L1 DNA sequence was modified, and all codons used in Pichia pastoris with high or highest frequency were used, and the formation of secondary structure and the selection of enzyme cutting sites were considered, and finally the HPV58 of the present invention was obtained Nucleotide sequence (SEQ ID NO: 1, figure 2 ).
Embodiment 2
[0042] Embodiment 2: Construction of HPV58 L1 recombinant expression vector
[0043] Synthesize the HPV58 L1 gene sequence of the present invention shown in SEQ ID NO: 1 and carry out PCR amplification as the template of PCR amplification, and it is cloned into pPICZαB vector (the described vector is purchased from Invitrogen, catalog number: V195-20).
[0044] Firstly, the 58L1 DNA fragment with BstBI and KpnI at both ends was amplified by PCR.
[0045] PCR primers:
[0046] Forward primer: 5'-ACTAATTA TTCGAA ACGATGTCTGTTTGGAGAC-3' (the underlined sequence is the BstBI restriction site);
[0047] Reverse primer: 5'-AGC GGTACC CTATTACTTCTTAACC-3' (the underlined sequence is the KpnI restriction site).
[0048] PCR reaction system:
[0049] Reactant
Volume (μL)
wxya 2 o
40.5
Forward primer (20pmol)
1
Reverse primer (20pmol)
1
10*buffer (with Mg2+)
5
dNTP (10mM each, Takara)
1.5
DNA t...
Embodiment 3
[0052] Example 3: Construction and expression of HPV58 L1 recombinant expression strain
[0053] The pPICZ58L1 vector was linearized with Sad. After the digestion reaction, the protein was removed with a phenol:chloroform (1:1) solution, and then 2.5 times the volume of absolute ethanol and 1 / 10 volume of 3M NaAc (pH 5.2) solution were added to precipitate the DNA. The resulting precipitate was washed with 75% ethanol, dried and washed with a small amount of sterile ddH 2 O dissolved the precipitate, electroporated into Pichia pastoris X-33 strain (Invitrogen), and spread on YPDS plate (1% Trypton, 2% Peptone, 2% glucose, 1M sorbitol) (containing 200 μg / ml Zeocin), 30 Cultivate for 3 days to obtain hundreds of clones. Dozens of clones were picked and inoculated on YPD plates (1% Trypton, 2% Peptone, 2% glucose, 1.5% agar powder) (containing 1500 μg / ml Zeocin) for screening high-copy plasmid strains, and cultured at 30°C for 2 days. Some clones grew faster, and several clone...
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