Recombinant antibacterial peptide TrSub as well as preparation method and application thereof
A technology of antimicrobial peptides and recombinant strains, applied in the field of genetic engineering and biology, can solve the problem that foreign proteins cannot be directly used as feed additives, and achieve the effects of low acid resistance and pepsin resistance, good thermal stability, and strong antibacterial activity
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Embodiment 1
[0026] Example 1 Error-prone PCR of recombinant antimicrobial peptide TrSub gene
[0027] Using the amino acid sequence of the antimicrobial peptide Sublancin168 published in GenBank: ACE07988.1 as the original sequence, it was translated into a coded nucleotide sequence using DNAMAN software, and the nucleotide fragment was artificially synthesized. Perform an error-prone PCR reaction on the artificially synthesized fragment, and the PCR amplification enzyme used is PrimeSTAR ® High-fidelity amplification enzyme, PCR reaction system (50 μL): 5 × PrimeSTAR ® Buffer 10 μL; dNTP 4 μL; PrimeSTAR ® 0.5 μL; pf 2 μL; pr2 μL; synthetic template 1 μL; ddH 2 O 30.5 μL. The PCR reaction conditions were: 95°C for 3 min pre-denaturation; 95°C for 30 sec denaturation, 56°C for 30 sec annealing, 72°C for 1 min extension, 35 cycles; 72°C for 10 min total extension; 4°C. After the PCR reaction was completed, the gene amplification target band of the antimicrobial peptide TrSub was detecte...
Embodiment 2
[0028] Example 2 The error-prone PCR product of the antimicrobial peptide Sublancin168 gene was inserted into the Trichoderma Tu6 expression vector to construct the recombinant expression vector Sub168-PCBHG
[0029] Use the linearization primer pair F: 5'-gctccgtggcgaaagcct-3' and R: 5'-agcacgagctgtggccaag-3' of the Trichoderma Tu6 vector to linearize it by PCR reaction, and the enzyme used in PCR is Phanta ® Super-Fidelity DNA Polymerase, PCR reaction system (50 μL): 5 × SF Buffer 25 μL; dNTP 1 μL; Phanta ® 0.5 μL; pf 2 μL; pr 2 μL; carrier PCBHG 1 μL; ddH 2 0 μL; 18.5 μL. The PCR reaction conditions were: 95°C for 3 min pre-denaturation; 95°C for 30 sec denaturation, 58°C for 30 sec annealing, 72°C for 8 min extension, 35 cycles; 72°C for 10 min total extension; 4°C. After the nucleic acid electrophoresis test, the template was digested and purified using the Cycle Pure Kit PCR purification kit to obtain the linearized vector recovery fragment. Ligate the fragment recov...
Embodiment 3
[0030] The transformation of the Trichoderma recombinant expression vector of embodiment 3 antimicrobial peptide TrSub
[0031]The Trichoderma reesei host strain was subcultured on a PDA+U solid medium plate under the condition of 30° C. for 5-6 days. The mature Trichoderma reesei host strain was inoculated into YEG+U medium, and cultured at 30 °C and 180 rpm for 20 h as the expression host. Filter and cultivate the mature bacterial liquid, collect the mycelium on the filter cloth into a sterilized Erlenmeyer flask, add lyase, and lyse it at 30°C and 90 rpm for 2 hours, and perform aseptic work at intervals of 30 minutes during this period. Take samples from Taichung, and use a hemocytometer to observe the morphology and number of lysed protoplasts under a microscope until reaching 10 8 Lysis was stopped at CFU / mL. The lysate in the Erlenmeyer flask was filtered and centrifuged, washed repeatedly with 1M sorbitol solution and resuspended 3 times to collect the final protopl...
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