The Screening Of A High Affinity Mutant Of MIF Receptor By DNA Shuffling

Macrophage-inhibitory factor (MIF) is one of the original cell factors ever described, and high level MIF may lead to sepsis, glucocorticosteroid rivalry, rheumatoid arthritis, tumors and so on. Some researches have demonstrated that sepsis mouses treated with anti-MIF monoclonal antibody could eacape from dying. This experiment indicated that diseases caused by high level MIF could be cured with an MIF inhibitor. Although antibodies always act as inhibitors, the anti-MIF monoclonal antibody is hard to prepare due to its high conservatism between mouse and human. And the humanization of antibodies is very difficult. A recent research by Ling N et al indicated that the outer-membrane region of CD74 (soluble CD74, sCD74), the chaperone molecule of MHC II, was the cell receptor of MIF, and the signal pathway initialled by MIF may be blocked by an anti-CD74 monoclonal antibody. The discovery of MIF receptor gives us another choice besides antibodies, that is, competitive binding of MIF by high affinity mutant of the receptor. To prepare a high affinity mutant of CD74, the technique of DNA shuffling is a simple and high efficiency choice. Many molecules have been evolved by this technique, bearing new characters of higher affinity, higher catalytic activity or higher stability. The present research aimed to obtain a high-MIF binding CD74 mutant through the technique of DNA shuffling and T7 phage display on the base of this finding, and to test the efficacy of this mutant in vitro. This research will contribute to the study of finding a clinic effective MIF inhibitor.Experiments and Results1. Preparation and verification of recombinant sCD74(1) Construction of sCD74-pQE-80L vector: First, Primers with BamH I or Sal I restriction enzyme sites respectively were designed according to the mRNA sequence BT019505. After the human sCD74 DNA cloned from the cDNA prepared by RT-PCR using the total RNA of white cells in peripheral blood, PCR products were analyzed by the agarose gel electrophoresis and DNA fragments of about 500 bp were recovered. Next, the purified sCD74 DNA was cloned into the pQE-80L vector and sequenced. Sequencing result indicated that the gene fragment of sCD74 was correctly cloned.(2) Overexpression and purification of recombinant sCD74: The recombinant sCD74-pQE-80L vector was transformed into the M15 competent cells and several single strain clones were picked out to be tested by restrict enzyme digestion. Then the positive M15 clone was inoculated into the Amp+, Kana+ LB medium and amplified until the OD600 value reached 0.6 before IPTG was added. After 4 hours the cells were collected and lysised by the supersonic method. And recombinant sCD74 in the supernatant was purified by the His affinity chromatography procedure and fractions were collected and analyzed by SDS-PAGE. The recombinant sCD74 protein with a purity of 95% analyzed by SDS-PAGE was prepared in the experiments previously done.(3) Verification of recombinant sCD74: The recombinant sCD74 was transferred onto a PVDF membrane and analyzed by WB using the anti-CD74 monoclonal antibody, LN-2. This antibody was also used in the ELISA experiment to test the binding of recombinant sCD74 and MIF. Briefly, the recombinant sCD74 was added into a 96 well ELISA plate pre-coated with MIF and LN-2 antibody and goat anti-IgG antibody were added subsequently into the wells with the dilutions of 1:1,000 and 1:2,500 respectively. And the result was tested at 495 nm 15 min after the OPD reagent was added. The recombinant sCD74 was proved to be right by the western blot analysis. And the OD values showed good linearity with multiple diluted recombinant sCD74 (R2=0.9925), which proved the report that CD74 may bind with CD74 with high affinity.2. DNA shuffling of the sCD74 gene(1) Cloning of homo sCD74 genes: The genes of Mouse (NM₀10545) and Bos taurus (BT021489) were aligned with that of human sCD74, and the homologous regions were found out for primer design. Then the sCD74 DNA fragments from the three origins were cloned from the cDNA of white blood cells in the peripheral circulation and sub-cloned into the pGEM-T vector, and positive clones by restriction enzyme digestion were sent to Invitrogen Ltd. to be sequenced. Sequencing results indicated that the gene fragment of homologous human, mouse and bos taurus were correctly cloned.(2) Site directed mutagenesis: Primers for the overlapping PCR to make samesense mutations in the Hind III enzyme sites of the homologous sCD74 were designed with the Hind III enzyme site mutated into AAgCTg from AAgCTT. Mutants were sub-cloned into the pGEM-T vector, and positive clones by restriction enzyme digestion analysis were sent to Invitrogen Ltd. to be sequenced. Sequencing results indicated that the Hind III enzyme sites were successfluly mutated.(3) Preparation of short random DNA fragments: Equal amounts of the homologous sCD74 DNA fragments were mixed together and digested with DNase I for 30 min at 37℃. Digestion products were analyzed by the agarose gel electrophoresis and 30-50 bp DNA fragments were recovered by the High Efficiency Little DNA Recover Kit (type B).(4) DNA shuffling: Firstly, a primerless PCR was carried out. To perform this experiment, dNTP, Mg2+, taq DNA polymerase and short random DNA fragments were added in a 200μl microtube, and 20 cycles of PCR reaction were taken. Products of the primerless PCR were used to perform a secondary PCR reaction by adding specific primers with EcoR I or Hind III restriction enzyme site. Products of both the primerless PCR and the secondary PCR were analyzed by a 1.0% agarose gel electrophoresis, and the DNA fragments of about 500 bp were recovered.3. Construction and screening of the T7 phage library(1) Construction of the T7 phage library: The 500 bp DNA fragments recovered previously were digested by the EcoR I and Hind III restriction enzymes for 2 hours and purified by the agarose gel electrophoresis. Then the digested DNA fragments were ligated with the T7Select 10-3 EcoR I/Hind III Vector Arm, which was provided in the T7Select?10-3b Cloning Kit (Novagen), at the ratio of 3:1. After over night ligation at 16℃, 5μl ligation products were added totally into 25μl T7 Packaging Extracts provided in the T7Select?biopanning kit (Novagen) and the original phage library was constructed. 5μl liquid from the original phage library was took to test the library capacity. A library with the capacity of 2.1×107 was generated.(2) Screening of the T7 phage library. The recombinant MIF was coated 0.5μg per well in a 96-well ELISA plate over night and the plate was blocked with the blocking reagent for 2 hours at 37℃. After the plate washed by 1×TBS, phages were added into the ELISA plate and incubated at room temperature for 30 min. After washing with 1×TBST for 3 times, the Elution Buffer was added to elute the phages still binding with MIF. Then the eluted phages were amplified by affecting the BLT5403 strain. The phage forming units (pfu) of the amplified and un-amplified eluted phage were tested. In the following 3 rounds of screening, recombinant sCD74 was used to block MIF before the addition of phages. Gradually reduced incubation time and improved elution time were taken and the elution condition was stricter round by round. Phages were efficiently enriched, with the enrich index of 3.3×10-5 and 200 clones eluted finally after 4 rounds of biopanning.(3) Verification of the high affinity phage mutant: Phage dilutions of different folds from the forth round screening were used to form plaques and the plate with 100 plaques was taken to carry out subsequent experiments. 40 plaques from this plate were picked out to test the positive rate by PCR analysis. Improvement of MIF binding affinity of positive phages was tested by a competitive ELISA and inserted genes in 3 random picked high affinity phage clones were cloned and sequenced by Invitrogen Ltd. The high MIF binding phage, PHCD74mu, was found to be capable to inhibit sCD74 from binding to MIF and the OD values shows linearity with the concentration of PHCD74mu (R2=0.9905). Three phage clones randomly picked was found to be a same clone harboring a gene fragment encoding a 31 aa peptide. This mutant was named HCD74mu.4. Overexpression, purification and characterization of high affinity sCD74 mutant(1) Construction of HCD74mu-pET42a vectors: HCD74mu DNA was cloned into the EcoR I and Hind III enzyme sites of the pET42a vector and the correct recombinant HCD74mu-pET42a vector was transformed into the BL21 competent cells. The positive single HCD74mu-pET42a clone by enzyme digestion analysis was sent to Invitrogen Ltd. to be sequenced. Sequencing result indicated that the HCD74mu gene was successfully cloned into the pQE-80L vector.(2) Overexpression and purification of HCD74mu: The BL21 single clone harboring the correct recombinant HCD74mu-pET42a vector was amplified and induced, and cells were lysised by the supersonic lysis method. The recombinant HCD74mu in the supernatant was purified by a GST affinity chromatography column and analyzed by SDS-PAGE. The purified protein was found to have the purity of 95% according to the SDS-PAGE result.(3) Evaluation of the sCD74 inhibiting ability by the recombinant HCD74mu: A competitive ELISA was carried out in a 96-well ELISA plate, and the ability to inhibit sCD74 from binding with MIF revealed by the recombinant HCD74mu was evaluated. HCD74mu was found to be capable of inhibiting sCD74 from binding with MIF by the competitive ELISA (p<0.01) and the OD values showed good linearity with HCD74mu concentrations (R2=0.9969).ConclusionThe present research constructed a phage library of shuffled sCD74 DNA using homologous sCD74 fragments of human, mouse and bos taurus, and a high MIF binding mutant, HCD74mu, has been obtained. The HCD74mu gene was sub-cloned into the pET42a vector and overexpressed in the BL21 strain. And the purified recombinant HCD74mu was found to have the ability to inhibit sCD74 from binding with MIF by competitive ELISA. This research made an attempt to find a new way to cure diseases using high affinity mutants of receptors, and this research will also contribute to the study to find a clinic effective MIF inhibitor.