A kind of site-directed mutagenesis lambda phage int recombinant protein and preparation method thereof
A recombinant protein and site-directed mutation technology, applied in the biological field, can solve problems such as limiting high-throughput vector construction methods, affecting enzyme recombination activity, and being unfavorable for large-scale recombination reactions
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0039] Embodiment 1: clone wild-type and mutant Int protein gene
[0040] 1) Clone the lambda phage Int protein gene with site-directed mutation.
[0041] 2) Design primers according to the Lambda phage Int protein gene sequence published on NCBI, as described in the following table:
[0042]
[0043] 3) Use the designed upstream and downstream primers of Int-wt and Int-(G347H)-(D351H) to perform PCR reaction system as follows:
[0044] reaction system:
[0045]
[0046]
[0047] 4) PCR reaction conditions are as follows:
[0048]
[0049] 5) PCR product recovery
[0050] After the PCR, the length of the fragment was analyzed by 2.0% agarose gel electrophoresis, the target band was cut out according to the size of the fragment, and the gel-cut product was recovered using Sangon’s DNA purification kit.
Embodiment 2
[0051] Example 2: Construction of wild-type and mutant expression strains
[0052] Construct the site-directed mutation gene into the expression plasmid, and obtain the positive expression strain
[0053] 1) Construct the lambda phage Int protein gene with site-directed mutation containing restriction sites, select the Int-full-length upstream and downstream primers, and use the PCR product obtained in Example 1 as a template, and the double enzyme digestion system of the plasmid and PCR product is as follows:
[0054] Double digestion reaction of PCR product and plasmid vector
[0055] Enzyme digestion system for PCR products:
[0056]
[0057]
[0058] Reaction conditions: 37°C for 4 hours
[0059] Plasmid vector digestion system:
[0060]
[0061] Reaction conditions: 37°C for 4 hours
[0062] The PCR product and the vector digested product were subjected to 1% agarose gel electrophoresis, and were purified and recovered using a DNA gel recovery kit.
[0063] ...
Embodiment 3
[0086] Embodiment 3: attB site PCR product obtains
[0087] 1) Construct a PCR product containing attB1 and attB2 sites.
[0088] 2) Design a pair of primers containing attB1 and attB2 sites with reference to the human NFKBIA gene sequence (NCBINo.NM_020529.2). The details are shown in the following table:
[0089]
[0090] 3) Use the designed attB site upstream primer (attB1 site) and downstream primer (attB2 site) for PCR reaction system as follows:
[0091] reaction system:
[0092]
[0093]
[0094] 4) PCR reaction conditions are as follows:
[0095]
[0096] 5) PCR product recovery
[0097] After the PCR, the length of the fragment was analyzed by 2.0% agarose gel electrophoresis, the target band was cut out according to the size of the fragment, and the gel-cut product was recovered using Sangon’s DNA purification kit.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More