Genetic engineering immunosuppressive polypeptide and preparation method and application
An immunosuppressive and genetic engineering technology, applied in the fields of genetic engineering immunosuppressive polypeptides, preparation and use, can solve the problems of poor potassium channel selectivity, toxic and side effects, etc., and achieve the effects of strong specificity, high stability, and not easy to deteriorate.
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Embodiment 1
[0050] Molecular design of embodiment 1 LWX-1 and LWX-AD-1 polypeptide sequence
[0051] Through protein structure prediction ( http: / / swissmodel.expasy.org / / SWISS-MODEL.html ) SWISS-MODEL program and protein structure analysis ( http: / / swissmodel.expasy.org / spdbv / ) Swiss-Pdb Viewer program, based on our deep understanding of the diversity and mechanism of interactions between scorpion venom polypeptides and potassium channels (Biophysical Journal, 2004, 87: 105; J.Proteome.Res.2007, 6: 611; J .Chem.Inf.Model., 2007, 47 (5): 1967; Proteins-In press), molecular design specifically acts on the protein polypeptide sequence of potassium channel Kv1.3:
[0052] LWX-1: VGINVKC (1) KHSRQC (2) LKPC (3) KDAGMRFGKC (4) TNGKC (5) HC (6) TPK (SEQ ID NO: 1)
[0053] LWX-AD-1: NEAVPTGGC (1) PFSDFFC (2) AKRC (3) KDMKFGNTGRC (4) TGPNKTVC (5) KC (6) SI (SEQ ID NO: 2)
[0054] Both LWX-1 and LWX-AD-1 polypeptides have 3 pairs of disulfide bonds in the molecule, and their pairing methods ar...
Embodiment 2
[0055] Example 2 Expression, purification and analysis of LWX-1 and LWX-AD-1 polypeptide sequences
[0056] According to the LWX-1 and LWX-AD-1 polypeptide sequences, the nucleotide sequence encoding the protein was deduced by conventional methods, and on this basis, primers were designed for PCR amplification to obtain the target gene.
[0057] 4 primers for LWX-1 polypeptide:
[0058] Forward primers are:
[0059] Primer P1: 5'GTGAATTCGATGACGATGACAAGGTGGGTATTAATGTGAA3';
[0060] Primer P2: 5'GGTATTAATGTGAAATGTAAGCATAGCCGCCAATGTCTGAAA CCATGCAAGGATGCTG3';
[0061] The reverse primer is:
[0062] Primer P3: 5'GGTACAATGGCATTTGCCATTGGTGCATTTACCAAATCTCATT CCAGCATCCTTG3';
[0063] Primer P4: 5'TAGCTCGAGTCACGGGGTACAATGGCATTTGC3'.
[0064] 2 primers for LWX-AD-1 polypeptide:
[0065]Primer P1: 5'GATGGATCCGATGACGATGACAAGAATGAAGCGGTGCCAACA3'
[0066] Primer P2: 5'GCTCTCGAGTTAGATAGAACATTTACAAAC3'
[0067] For the LWX-1 polypeptide, two rounds of PCR amplification reactions were ...
Embodiment 3
[0081] Example 3 Analysis of the Pharmacological Activity of Recombinant LWX-1 and LWX-AD-1 Proteins on Potassium Channel Kv1.3 Targets
[0082] COS-7 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2, and the potassium channel Kv1.3 recombinant plasmid was treated with Sofast TM The transfection kit was used for transfection, and the transfected cells were selectively cultured on 0.8mg / ml Geneticin medium. The pharmacological activities of LWX-1 and LWX-AD-1 proteins were determined and analyzed using a whole-cell patch clamp instrument (EPC-10 dual-channel patch clamp amplifier HEKA, Elektronik, Lambrecht, Germany). The setting of experimental parameters, data collection and stimulation application are all controlled by Pulse software. The filter 1 of the instrument is set to 10kHz (Bessel), the filter 2 is set to 2.9kHz (Bessel), and the electrode impedance is 2-5MΩ. After forming a high-impedance (1-5GΩ) seal between the electrode and...
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