Procambrus clarkii type i lysozyme gene, lysozyme coded by lysozyme gene and application of lysozyme
A technology of Procambarus clarkii and lysozyme, which is applied in the field of genetic engineering, can solve problems such as the absence of Procambarus clarkii lysozyme gene
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Embodiment 1
[0077] Embodiment 1: the cloning of cDNA of lysozyme gene cDNA of Crayfish clarkii type I
[0078] 1) Extraction of total RNA: Total RNA was extracted by one-step method using the prior art.
[0079] 2) cDNA first-strand synthesis: 4 microliters of total RNA, plus 1 microliter of SmartF (5'-TAC GGC TGC GAG AAG ACG ACAGAA GGG-3') and 1 microliter of Oligoanchor R (5'-GAC CAC GCG TAT CGA TGT CGA CT 16 (A / C / G)-3'), react at 72°C for 5 minutes, then add 4 microliters of 5-fold Buffer, 1.25 microliters of dNTP, 0.625 microliters of RNase inhibitor, 1 microliter of MMLV reverse transcriptase, RNase-free 12.875 microliters of sterilized water were reacted at 42°C for 60 minutes, and the reaction was terminated at 70°C for 10 minutes.
[0080] 3) PCR reaction: chain polymerase reaction (PCR) reagents and conditions:
[0081] First mix the following reagents together:
[0082] 5 microliters (μl) of 10xTaq DNA polymerase buffer
[0083] ·Template cDNA 1μl
[0084] ·Forward primer ...
Embodiment 2
[0104] Example 2: Construction, expression and purification of prokaryotic recombinant expression vectors
[0105] (1) According to the sequence of Procambarus clarkii lysozyme (remove the signal peptide sequence) and the cloning site of the expression vector pPET30a (Novagen Company), the present invention has selected the BamH I and Xho I restriction sites of the pET30a cloning site, Therefore, when designing the primers, a BamH I restriction site was introduced into the upstream primer, and an Xho I restriction site was introduced into the downstream primer.
[0106] (2) Gene amplification, cloning and recombinant plasmid screening
[0107] Using pMD-18T-Lysi as a template, the PCR reaction was carried out with the designed expression primers. The amplification conditions were: 94°C, 2min pre-denaturation; 94°C, 30s, 56°C, 45s, 72°C, 45s, 35 cycles; 72 ℃ extension 10min.
[0108] 2% agarose gel electrophoresis PCR product detection:
[0109] The PCR product was subjected...
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