Construction And Expression Of Wild-type And Mutant MBL In CHO Cells

Background:Mannose-binding lectin (MBL) is the key molecular in innate immune system. It is secreted by liver and mainly exists in human plasma. The concentration of MBL in plasma is about 0-8.24μg/ml, while higher than 1μg/ml is very important for its biological function. The concentration lower than 0.5μg/ml is called MBL deficiency. Six gene mutations, has been found to be responsible for the lower concentration of MBL. And exon 154,57,52 which encode the collagen-like domain are especially important. Besides single nucleotide polymorphisms(SNP) in promoter-550(H/L),-221(X/Y), and 5'untranslated region+4 (P/Q) can also influence the concentration of MBL. Codon 54 mutation are more common in Caucasoids, Chinese, and Eskimos (gene frequencies 0.52), Codon 57 mutation is more common in Africans (gene frequencies 0.50-0.60), while codon 52 mutation is in low frequency in Caucasoids and Africans (gene frequencies 0.14). Only codon 54 mutation is found in Chinese at present. Previous studies have indicated that MBL deficiency is associated with the common opsonic defect and the susceptibility to infections, especially in immune compromised individuals. MBL is usually made up of two to six subunits, each subunit consists of three identical 32-KDa polypeptide chains which contain a N-terminal cysteine-rich region, a collagen-like domain, a "neck" region and a carbohydrate-recognition domain (CRD). The CRD of MBL could recognize and bind to mannose, fucose and N-acetylglucosamine structures on the surface of bacterium, virus, fungi, parasite and so on. MBL could inhibit influenza A virus, HIV, Ebola virus, Marburg virus in vitro. But whether MBL could inhibit HCMV in vitro hasn't been reported now. In this study, expression system of wild-type and codon 54 mutant mannose-binding lectin (MBL) in vitro were constructed, and rMBL of wild-type was acquired and characterized. For the further study about MBL inhibit HCMV in vitro and possible future clinical applications for replacement therapy.Objective:Construction and expression of wild-type and codon 54 mutant human mannan-binding lectin (MBL) in vitro, purify and characterize the recombinant MBL, For the further study about MBL inhibit HCMV in vitroMethods:The MBL expression plasmids PIRES2-AcGFP-MBL and PIRES2-AcGFP-MBLm54 were constructed and transfected into Chinese hamster ovary (CHO-K1) cells. G418 was added for screening. At 24h,48h and 1 week after transfection, MBL gene expression of the wild-type and codon 54 mutant were detected by fluorescence microscopy. The level of MBL mRNAs were quantified by real time polymerase chain reaction. The target gene expression in transfected CHO cells were more than 103 times higher than the negative control. The mannose-binding activity of recombinant human MBL were determined by ELISA procedure. The wild-type rMBL was purified from the culture supernatant by affinity chromatography on mannan-Sepharose 4B. The concentration of recombinant MBL were determined by Bradford protein assay. And its molecular weight and purity were analyzed by SDS-PAGE under nonreducing and reducing condition.Results:1. Eukaryotic expression plasmids PIRES2-AcGFP-MBL and PIRES2-AcGFP-MBLm54 were constructed successfully.2. Eukaryotic expression plasmids PIRES2-AcGFP-MBL and PIRES2-AcGFP-MBLm54 were transfected into CHO cells. Observed by fluorescence microscopy and quantified by real time PCR, wild-type and codon 54 mutant MBL genes were expressed in CHO cells successfully.3. The wild-type recombinant MBL had sugar specificities. The codon 54 mutant MBL had little mannose-binding activity by ELISA.4. The quantities of purified MBL were determined by Bradford protein assay. And the concentration of rMBL in CHO-MBL culture supernatant was 559.82 ng/ml after three days of culture.5. The purified MBL was analyzed by SDS-PAGE,32KDa component in the purified recombinant product was found in reducing condition and component larger than 170 KDa was found in unreducing condition.Conclusions:1.The eukaryotic expression plasmids of the wild-type and codon 54 mutant MBL gene were constructed and expressed in CHO cells successfully.2.Codon 54 mutant led to the low expression of oligomer and polymer MBL in the CHO culture supernatant.3. Recombinant wild-type MBL was of high purity and was oligomer and polymer protein which owned mannose-binding activity. The concentration of recombinant MBL in culture supernatant was high enough for the further study.This work was part of the project:the mechanisms of MBL inhibit HCMV invading the target cells in vitro, and it provided the basis for further study and possible future clinical applications for replacement therapy.