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The Screening of a High Affinity Mutant of MIF Receptor by DNA Shuffling

Author: ZuoJiaTao
Tutor: HuChuanMin
School: Third Military Medical University
Course: Clinical Laboratory Science
Keywords: CD74 MIF DNA shuffling DNA shuffling T7 phage display
CLC: R91
Type: Master's thesis
Year: 2008
Downloads: 113
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Abstract


Macrophage inhibitory factor (Macrophage-inhibitory factor, MIF) is one of the earliest elaborated cytokines Previous studies have shown that the MIF high expression is induced sepsis, glucocorticoid antagonism, rheumatoid arthritis, tumor The main reason of the pathological process. Researchers to anti-MIF monoclonal antibody treatment septic mice found that can significantly increase the survival rate, suggesting that blocking MIF can inhibit the pathological process caused by the high MIF expression. But high MIF conservative sexual antibody preparation difficulties; antibodies into clinical applications, the source of the problem is a major obstacle. Recent studies found that the extracellular domain of CD74 (MHC II invariant chain accessory molecules) 73-232 aa (sCD74) natural receptors for MIF blocking MIF and CD74 binding can block the signaling pathways mediated by MIF. The MIF receptor found for the inhibition of MIF high expression to provide a new choice - receptor competition. The DNA shuffling' dissertation">DNA shuffling technique is a simple and efficient protein in vitro evolution techniques, there are many molecules through DNA shuffling has a higher affinity, higher catalytic activity or better stability. This study using DNA shuffling technology (DNA shuffling) of sCD74 genes restructuring, combined with T7 phage display technology (T7 phage the display), screening affinity binding of MIF mutant plant height, and preliminary identification, is looking for a MIF inhibitor lay the foundation for potential clinical prospects. Main research content and results 1.sCD74 prokaryotic expression and verification (1) sCD74-pQE-80L recombinant vector construct prokaryotic expression and purification: the Genebank query CD74 mRNA sequence (BT019505) as a template, the design containing BamH I Sal I restriction sites on the downstream-specific primers, from human peripheral blood leukocyte cDNA clone sCD74 gene; objective fragment was purified after BamH I, Sal I restriction digestion, connecting the carrier of pQE-80L, restriction enzyme identified the evacuation Ivitrogen Company sequencing analysis; sequencing correct recombinant sCD74-pQE-80L plasmids were transformed into competent bacteria of M15 induced expression; bacteria disrupted by ultrasonic, and centrifuged supernatant His affinity chromatography purification, stars gradient elution, collected elution ingredients of SDS-PAGE analysis. Obtained through the above-mentioned experimental cloning human sCD74 gene prokaryotic expression products His affinity purified purity of about 95% determined by SDS-PAGE. (2) sCD74 and MIF combined verification: semi-dry transfer method of purified recombinant sCD74 transferred to PVDF membrane, mouse anti-human CD74 monoclonal antibody (LN-2) and sheep anti-mouse IgG western blot analysis, chemiluminescence The assay results; the MIF package is a 96-well ELISA plate, adding different concentrations of recombinant sCD74 add OPD chromogenic After incubation with mouse anti-human CD74 monoclonal antibodies (LN-2), and sheep anti-mouse IgG, 450 nm, OD value was measured. The prokaryotic expression sCD74 was verified by western blot correct; ELISA experiments show fold dilution sCD74 and MIF linearly, R2 = 0.9925, proved MIF and CD74 combined reported. The reorganization of sCD74 2.DNA shuffling gene (1) cloning of human, mouse, bovine sCD74 homologous genes: human, mouse (NM 0 10545), cattle (BT021489) CD74 cDNA sequence of biological bioinformatics analysis, to determine the person, mice, bovine sCD74 homologous region, specific primers were designed from the peripheral blood leukocyte cDNA templates cloned sCD74 homologous region DNA fragments; cloned three homologous gene DNA fragment was ligated promega the pGEM- T vector, blue-white screening, then the restriction endonuclease Invitrogen, cutting the identification of positive clones were sequenced. Sequencing results showed that the cloned gene fragments of human, mouse, bovine sCD74 correct. (2) the human, mouse, bovine sCD74 site-directed mutagenesis of the homologous gene: design directed mutagenesis primers staggered extension PCR mutated to human, mouse, bovine sCD74 homologous internal Hind III restriction sites (AAGCTT) the AAgCTg, mutations product connection promega company pGEM-T vector and transformed into DH5α competent blue-white screening, then the positive clones identified by restriction enzyme cutting sequencing analysis sent to Ivitrogen company. The sequencing results showed that the human, mouse, bovine sCD74 gene internal Hind III restriction sites have been directed mutagenesis AAgCTg. (3) Preparation of random small fragments of DNA: human, mouse, bovine a homologous the sCD74 DNA fragment, by equimolar ratio mixed, digested with DNase I 37 ℃ 30 min, 2.0% agarose gel electrophoresis analysis of the digestion product, cut 30 -50 bp size DNA fragments, the small DNA fragment of the type B efficient recovery kit (broad Tektronix) recovered. (4) DNA shuffling: First primer PCR, random small fragments of the recovered DNA was added to 200μl microcentrifuge tube, add mM dNTP, Taq enzyme and buffer, Mg2 ddH2O made up to 20μl, the reaction of 20 cycles at 2.0 % agarose gel electrophoresis analysis of the product; respectively, was added to the non-primer PCR product tube containing the EcoR I and Hind III restriction sites upstream and downstream primers specific press 50μl system supplemented with dNTP, Taq Enzyme buffer Mg2, etc., the reaction of 15 cycles, the product was analyzed in a 1.0% agarose gel electrophoresis, about 500 bp fragment was recovered under. 3.T7 phage display library construction and screening (1) T7 phage display library building: the recovery of the size of the 500 bp DNA fragment EcoR I, Hind III restriction enzyme cutting, recovered in a 3:1 molar ratio T7Select? 10-3b Cloning Kit (Novagen) kit T7Select 10-3 EcoR I / Hind III Vector Arm 16 ° C connection overnight; 5μl ligation products with 25 μl T7Select, T7 packaging extracts? biopanning kit (Novagen) kit The hybrid assembly phage (2 parallel tubes); take 5μl assembled product raw capacity of the determination of the phage library. Was determined to have been assembled by a capacity of 2.1 × 107 pfu of mutant original library. (2) panning method screening of high-affinity mutant phage: MIF 0.5μg / hole package 96-well ELISA plate, closed after the original phage added to 96-well plates (2 wells), room temperature for 30 min and discard hole liquid, to TBST washed 3 times, plus the the eluent hole bound phage to infect BLT5403 host bacteria, amplified to OD600 started to decline, the determination of amplification around phage titer (PFU); recombinant sCD74 closed MIF before adding phage, a gradual reduction of the mutant phage MIF role, at the same time gradually improve washing efforts to extend the elution time, and then for three panning. After four rounds of panning, phage effective enrichment of the fourth round of panning clone 200, enrichment value of 3.3 × 10-5. (3) the identification of the high affinity mutant bacteriophage: To establish the fourth round of panning the supernatant phage tablet, to select the number of plaques of about 100 / plate Flat 40 monoclonal randomly picked and amplified PCR identification positive rate; of the PCR-positive phage and prokaryotic expression of sCD74 mutant phage inhibitory effect of sCD74 combined MIF monoclonal competitive ELISA assay, PCR cloning plant height affinity strains contained the inserted fragment sent Ivitrogen sequenced. Competitive ELISA analysis showed high affinity mutant phage PHCD74mu vitro inhibit the binding effect of sCD74 and MIF (p LT; 0.01), linear (R2 = 0.9905) and the binding of phage with MIF; three randomly picked single The cloning and sequencing analysis showed that the same clone, named this mutant HCD74mu, and short peptide is a 31 amino acids in length. 4. A high affinity for sCD74 mutation body (HCD74mu) of the original nuclear expression, purification and identification (1) HCD74mu-pET42a expression vector construction: respectively contain, EcoR I, Hind III digested bit point of the upper, downstream primer cloning HCD74mu coding genes The double digested connection pET42a vector and transformed into E.coli BL21, PCR identification of positive monoclonal send Ivitrogen sequenced. The sequencing results showed that the cloned correct. (2) HCD74mu the prokaryotic expression and purification: correct sequencing HCD74mu-pET42a plasmid was transformed into BL21 competent bacteria were induced, collected by centrifugation bacteria sonication, centrifuged supernatant GST affinity chromatography purification, by SDS-PAGE Identification of the purified product. SDS-PAGE showed that the purity of the product is about 95%. (3) competitive inhibition ELISA recombinant HCD74mu effect of sCD74 and MIF: MIF package is a 96-well ELISA plate, competitive ELISA determination of recombinant expression HCD74mu inhibiting effect of sCD74 binding of MIF. The results show that the recombinant HCD74mu have significant in vitro competitive inhibition of the binding effect of sCD74 and MIF (p lt; 0.01) and different concentrations HCD74mu to linear (R2 = 0.9969), in conjunction with the MIF. Conclusion: In this study, human, mouse, cattle CD74 extracellular homologous region gene as a template, use a combination of DNA shuffling and T7 phage display technology to build a reorganization of sCD74 T7 phage display library after four rounds of screening has been a The high affinity binding MIF mutant HCD74mu; constructed HCD74mu the prokaryotic expression vector, inducing expression of GST affinity chromatography purification; by the competitive ELISA validation, HCD74mu having the ability to inhibit the binding of MIF and CD74 in vitro. Of this research is to find a clinical role of MIF inhibitor has laid the foundation, and to explore the possibility of a competition point of view from the receptor for the treatment of disease, the research is of great significance.

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