Expression Of Full-length MBL Gene Of Han Chinese In CHO Cells

Mannan-binding lectin(MBL) is one member of collectins in the C-type lectin superfamily.MBL appears in plasma as a series of oligomers from dimmers to hexamers,and its higher oligomers bind the mannan with high affinity and efficiently active the complement.The carbohydrate-recognition domain(CRD) of MBL can bind the specific saccharide structures in pathogen such as mannose, fucose,N-acetyl-D-glucosamine,N-acetyl-mannosamine,glucose,which make MBL be able to recognize various pathogens including streptococcus,staphylococcus aureus,Listeria monocytogenes,Neisseria meningitidis,chlamydia pneumoniae, influenza A,HIV,candida albicans and cryptosporidium parvum.It acts as a role of first defense in the way of complement activation and opsonophagocytosis.So MBL has been concidered as one of the most important pattern recognition moleculs.The low concentration and functional deficiency of serum MBL correlates with many diseases such as recurrent bacterial infections,recurrent spontaneous abortion, systemic lupus erythematosus,rheumatoid arthritis and so on.And the main etiopathogenisis is the mutation in the gene of MBL.It has been found that there are three missense point mutations in the exon 1 of MBL gene at codon 52,54 and 57 that respectively cause MBL deficiency.Such mutations in the genetype of MBL gene are correspondingly termed as type D,B and C,while the wild -type is A.These mutant genes are codominant inheritance on euchromosome and appear high frequency spreading in various ethnic population.So the replacement therapy to MBL deficiency patients is naturely required.There have been some reports of MBL therapy followed the accomplishment of phage I trial,which gave some reason for optimism.However,the MBL purified from plasma can't avoid certain disadvantages:the concentration of MBL in plasma is totally low and it requires a great quantity of plasma which is limited to the source of blood.Also it is not free from the risks of the viral contamination such as HIV and HCV.Additionally,due to the great complex of plasma,it is hard to exact the MBL with high purity which is required in the medicine research.So it is great significant to prepare sufficient recombinant MBL that is similar to plasma MBL in structure and function.Thus it is also important for research in the regulation of the expression of MBL gene.Our study group have cloned the MBL cDNA of Han Chinese and applied it to express in mammalian cells.However,the quantity and oligomers of recombinant protein is far-fetched.Considering that the uncoding region may regulate gene expression,we try to make expression of the full-length MBL gene.1.Cloning and sequence analysis of full-length MBL gene of Han Chinese.Detect the level of serum MBL by the measurement coated with mannan in plate and detected by the anti-MBL(HYB131-11).The blood samples from 8 healthy chinese without history of recurrent infection and autoallergic disease were diluted into 1:2,1:4,1:8 for detection.The results were analyzed by the statistical method of repeated measures using SPSS13.0.The sample with higher level of serum MBL was chose for extraction of genomic DNA from leucocytes.Taking the genomic DNA as template and applying the appropriate primers,the full-length MBL gene was amplified with Platinum Taq by the method of high fidelity PCR. Then the PCR products were subcloned into the vector pCR(?)-XL-TOPO by TA clone.After identification by endonuclease digestion and PCR,the constructed plasimds,pXL-MBL,were sent to Invitrogen for sequencing analysis.The results of ELISA showed that the decrease of detecion in OD_450nm with the increase in dilution indicated that the measurement was reliable(F=99.351,P＜0.001).And the values of OD_450nm were varied from different samples(F=27.468,P＜0.001).The higher one was chose for further extraction of genomic DNA.The sequencing result show that the MBL gene with a length of 6 321 bp containing the whole exons and introns is inserted into to the pCR(?)-XL-TOPO vector in inverse direction.Aligned with the MBL gene previously recorded in GenBank(NC₀00010.9 Region: 54195146～54201466),it's found that both of them are identical except 17 nucleotides,among which there is only one laying in the coding region of exon 4 and resulting in the change of 126th coden(CTC→CTG) but not the change of coding amino acid(leucine).And this one in exon 4 is even consistent with that of the MBL cDNA in GenBank(AH006353),while the others lay in the three introns and two uncoding regions of exons.The exact nucleotide sequence has been deposited in GenBank with the accession number EU596574.2.The construction of the eukaryotic expression plasmid of the full-length MBL geneAccording to the restriction map of the plasmids,pXL-MBL,pcDNA3.1(-) and pCI-neo,subcloned the MBL gene into new vectors by appropriate endonuclease digestion in order to construct recombinant eukaryotic expression plasmid.(1)The construcion of pcDNA3.1(-)-MBL:Digested respectively pXL-MBL and pcDNA3.1(-) both by Kpnâ… and Notâ… .Exact the target fragment and vector fragment after agarose gel electrophoresis.After ligation of such two fragemts with T4 ligase,transformed it into the Top10F' and selected the positive colonies with ampicilin for further plasmid exaction.Then the new constructed plasmid was identified by endonuclease digestion,PCR and sequence.The results show that the full-length A-type MBL gene is inserted into the vector of pcDNA3.1(-) in right direction and the pcDNA3.1(-)-MBL has been successfully constructed.(2) The construcion of pCI-MBL:The plasmid pXL-MBL were digested completely by both Mluâ… and Smaâ… and incompletely by Xhoâ… into 7 fragments (1 429bp,1 952bp,2 734bp,3 718bp,4 686bp,6 552bp and 8 404bp).The 6 552bp one was the target fragment which contained the full-length MBL gene with two adhesive end of Mluâ… and Xhoâ… .Extracted such fragment and linked it to the vector fragment from pCI-neo pre-digested by both Mluâ… and Xhoâ… .The next steps were similar to the above(1),and the results also show that the full-length A-type MBL gene is inserted into the vector of pCI-neo in right direction and the pCI-MBL has been successfully constructed.3.The expression of pcDNA3.1(-)-MBL in CHO.The pcDNA3.1(-)-MBL was linerized with Blgâ…¡and transfected into CHO by using electroperforation.After transfection for 36h,complete medium containing 800μg/ml of G418 was used for 9 days followed by 400μg/ml of G418 for another 12 days to cultivate the transfected cells.G418-resistant cells were expanded to cultivate and identified by RT-PCR.Meanwhile,proceeded to clone the G418-resistant cells and screened out the positive monoclonicities by the above measurement of ELISA.The datas of detection were analyzed by the statistical method of one-way ANOVA and multiple comparison of Dunnett.Set the positive expression cell as the one which was detected higher than the twice of negative control.And it was shown that the positive ones were the cells E10(P=0.001), F10(P=0.001),D8(P＜0.001).D8 was chose to expand cultivation and the supernatant was collected for purification of the recombinant protein that was carried out by the mannan-Sepharose 4B affinity chromatography column.Then the purificaiton product was identified by western-blot.The results show that the MBL gene has been successful expressed in CHO and the recombinant protein appeared as high oligomers as plasma-deritived MBL(pMBL),though the quantity of the product is still not high.It may be resulted from the lost of MBL gene when the transfected cells proliferated.It is necessary to do more times for cloning in order to screen out the monoclonicities which express rMBL stablely and highly and do further research in biological function.