Novel fungal immunomodulatory protein FIP-NHA with antineoplastic activity and gene thereof
A technology of FIP-NHA and immunoregulatory protein, applied in the field of genetic engineering, can solve the problems of high protein cost, difficulty and high consumption of natural FIPs
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Embodiment 1
[0051] Example 1 Screening and cloning of the fungal immunoregulatory protein FIP-NHA enzyme coding gene fip-nha
[0052] Fungal genes were screened from the NCBI Fungal Genome Database based on the published conserved sequences of fungal immunomodulatory proteins. A pair of primers FIP-nha-F (EcoRI 5'- GAA TTC GCT ACT ACC AAC G-3') and FIP-nha-R (XhoI 5'- CTC GAG TTA CTT CCA TTG GGC GAC-3'), the fungal immunomodulatory protein FIP-NHA gene derived from the fungus Nectria haematococca was cloned by gene synthesis. The results of DNA sequence analysis showed that the full length of FIP-NHA was 342bp, and its cDNA sequence As shown in SEQ ID NO.2.
Embodiment 2
[0053] Example 2 Preparation of recombinant fungal immunoregulatory protein FIP-NHA.
[0054] The expression vector pGEX-4T-1 was subjected to double enzyme digestion (EcoRI and XhoI), and at the same time, the synthetic gene encoding the fungal immunoregulatory protein FIP-NHA was double enzyme digested (EcoRI and XhoI) to cut out the mature fungal immunoregulatory protein FIP -The gene fragment of NHA was connected with the expression vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-FIP-nha containing the fungal immunoregulatory protein FIP-NHA gene of the fungus Nectriahaematococca and transform it into Escherichia coli Rosetta to obtain the recombinant strain ROS / GST-FIP- NHA.
[0055] Take the Ros / GST-FIP-NHA strain containing the recombinant plasmid, inoculate it in 200 mL of LB culture solution containing 0.1 mM IPTG and 50 μg / ml ampicillin, induce culture at 25°C and 250 rpm for 6 hours, and collect the bacteria by centrifugation. It was then sonicated and the ...
Embodiment 3
[0056] Example 3 Determination of Hemagglutination Activity of Recombinant Fungal Immunomodulatory Protein
[0057] (1) 2 mL of healthy human cells (type A, B, AB or O) and rabbit blood cells were centrifuged at 2000 rpm for 5 minutes at room temperature.
[0058] (2) Carefully suck out the plasma supernatant, collect the red blood cells at the bottom of the centrifuge tube, carefully wash with pH 7.2 10mM PBS to fully resuspend the blood cells, centrifuge at 2000rpm for 5 minutes at room temperature, and wash 5 times in total.
[0059] (3) Finally, the blood cells were suspended in 1.5% (V / V) PBS.
[0060] (4) Prepare 0.2% gelatin PBS solution.
[0061] (5) Accurately weigh 1 mg of the recombinant Ganoderma lucidum immunomodulatory protein, dissolve it in 1 mL of PBS, and adjust the sample concentration so that the final working concentration is 1, 2, 4, 8 and 16 μg / mL.
[0062] (6) The hemagglutination reaction solution is mixed with 25 μl blood cell suspension, 25 μL reco...
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