Antibiosis usage of a family of protein
A technology of protein and family, which is applied in the field of antibacterial application of protein family, which can solve the problems of unclear protein function and lack of protein antibacterial properties
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Embodiment 1
[0063] Embodiment 1 Preparation and detection of human Glyrichin gene
[0064] 1. Obtaining the complete ORF sequence of human Glyrichin gene
[0065] According to the murine Glyrichin gene, the following pair of degenerate primers were designed to amplify the human fetal liver cDNA library, and the complete ORF sequence of human Glyrichin totaling 240bp, and the amino acid sequence deduced from the ORF sequence were obtained:
[0066] 5' Primer Pa: 5-CGATGCCGGTGGCCGTGGGTCCCT-3
[0067] 3' Primer Pb: 5-TTAGCATCGTATGCCCATTCCA-3
[0068] The PCR amplification conditions were: 94°C, 4 minutes, 1 cycle; 94°C, 40 seconds, 60°C, 50 seconds, 72°C, 1 minute, 30 cycles; 72°C, 7 minutes, 1 cycle. The PCR product was purified by Winzard PCRpreps purification kit, recombined into pGEM-T vector with T4 ligase, and then transformed into Escherichia coli JM109 for sequencing identification.
[0069] 2. Northern blot analysis
[0070] Four human tumor cell lines HepG2, HeLa, ...
Embodiment 2
[0087] Example 2 h / mGlyrichin expressed endogenously
[0088] Growth Inhibition Test of Escherichia coli BL21
[0089] hGlyrichin without any tag (that is, not fused with any protein) was recombined between the EcoRI and XhoI sites of the prokaryotic expression vector pET-22b(+), transformed into Escherichia coli BL21, and positive clones were identified by enzyme digestion. Pick positive clones 1 and 8, inoculate them into AMP-resistant liquid LB medium, and shake overnight at 37°C; inoculate at a ratio of 1:100 the next day, continue to shake until the OD value is 0.03, and pour into each test tube Add IPTG to a final concentration of 0.5mM, and add a corresponding volume of PBS to the negative control group; shake the bacteria at 30°C and 250rpm, take out 1ml of the bacterial solution every 45 minutes to measure the OD600 value, and measure 20 times continuously. Take time as the horizontal axis and OD600 logarithmic value as the vertical a...
Embodiment 3
[0090] Example 3 Purification and bactericidal activity detection of free hGlyrichin expression product
[0091]Primers were designed according to the cDNA sequence of mouse Glyrichin, and Enterokinase restriction sites were introduced into the primers, and human fetal liver mRNA was used for RT-PCR amplification. Under the action of the enzyme, insert it into the pGEX4-4T2 prokaryotic expression vector digested with the same endonuclease, after transforming JM109 Escherichia coli, induce expression with IPTG for 5 hours in vitro, collect the cells; resuspend in PBS, freeze repeatedly Melt and lyse; centrifuge at 4°C and 12,000 rpm for 20 minutes, and take the supernatant for SDS-PAGE electrophoresis. After confirming that the GST-Glyrichin fusion protein is expressed in the supernatant, the free hGlyrichin protein that is purified with GST affinity column and cut with medium enzyme has bactericidal activity. GST-Glyrichin fusion protein was induced and expressed as abov...
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