Alpha-1, 3 galactosyl transferase fusion protein and preparation method thereof
A fusion protein and galactose-based technology, applied in the biological field, can solve the problems of environmental pollution, poor effect, time-consuming and labor-intensive, etc., and achieve the effect of good industrial application prospects
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Embodiment 1
[0035] Cloning of the α-1,3-galactosyltransferase gene
[0036] Using methods known to those skilled in the art, the α-1,3 galactosyltransferase gene sequence that we cloned is shown in SEQ ID No.1, the corresponding α-1,3 galactosyltransferase gene sequence The amino acid sequence of the transferase is shown in SEQ ID No.2. For the convenience of subsequent cloning, a restriction endonuclease site such as NdeI was added upstream of the gene, and a BamHI site was added downstream of the gene.
Embodiment 2
[0038] Construction of α-1,3-galactosyltransferase expression clone
[0039] The α-1,3 galactosyltransferase gene cloned in the above Example 1 was introduced into restriction endonuclease NdeI and BamHI restriction endonuclease sites by PCR method, and the upstream primer sequence used was: 5'GAATACCATATGGAAAGCAAGCTTAAGCTATCG3' ; The downstream primer sequence is: 5'GAAGGATCCTTATCAGACATTATTTCTAAC3'. The recovered PCR product and pET-15b plasmid vector were digested with NdeI (NEB) and BamHI (NEB) restriction endonucleases for 3 hours; the vector was recovered after agarose gel electrophoresis to detect the digestion effect and Gene fragments; under the action of T4DNA ligase (NEB), connect the vector and the gene; add the ligated product directly to the competent E. coli DH5α melted in ice bath, incubate for 30 minutes and then heat shock transformation in 42°C water bath; The finally screened transformants were sequenced to verify the expression clone of α-1,3 galactosyltra...
Embodiment 3
[0041] Construction of expression clones fused with α-1,3 galactosyltransferase and TRX
[0042]The α-1,3-galactosyltransferase gene cloned in the above-mentioned Example 1 was introduced into restriction endonucleases KpnI and XhoI restriction sites by PCR, and the upstream primer sequences used were:
[0043] 5'GAATACGGTACCGAAAGCAAGCTTAAGCTATCG3' (sequence shown in SEQ ID No.4);
[0044] The downstream primer sequences are:
[0045] 5'GAACTCGAGTTATCAGACATTATTTCTAAC3' (sequence shown in SEQ ID No.4);
[0046] Both the recovered PCR product and the pET-32a plasmid vector were digested with KpnI and XhoI restriction endonucleases for 3 hours; the vector and gene were recovered after detecting the digestion effect by agarose gel electrophoresis; ligated in T4DNA Under the action of the enzyme (NEB), the vector and the gene were connected; the ligation product was directly added to the competent E. coli DH5α melted in an ice bath, incubated for 30 minutes and then transformed b...
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