Expressions Of Mutant MBL Gene In Eukaryotic Cells And Wild-type MBL Gene In Prokaryotic Cells

Mannan-binding lectin (MBL) is a serum protein belonging to the collectin family. On binding to specific carbohydrate structures on the surface of a broad range of microorganisms,MBL can activate complement system through the activation of MBL-associated serine proteases (MASPs) in antibody- and Clq-independent way. Activation of the complement may lead to killing of the microorganisms by direct lysis effect of complement system or the phagocytosis by the enhancement of the attachment of microbes to the phagocyte (opsonization). Low serun concentrations of MBL in man is the basis for a common opsonic defect associated with recurrent infection. MBL deficiency and low levels of serum MBL are strongly associated with the presence of variant MBL genes,encoding three different structural variants of the MBL polypeptide,which are caused by three point mutations,CGT52TGT,GGC54GAC and GGA57GAA,of MBL gene. The variant alleles are very frequent in populations,with the hightest frequence,0.29,found in Africas. The GGC54GAC allele is common in Chinese with gene frequency above 0.1. Hence,MBL deficiency is the most frequent immunodeficiency,but its pathological mechanisms remain elusive and await further investigation.The major research work in our laboratory is about the MBL deficiency,especially its molecular mechanism. There are two problems we need toelucidate-how the mutations of MBL gene lead to low level of MBL andif the function of mutated MBL proteins change?To construct eukaryotic expression vector of MBL gene with codon 54 point mutation,the target sequence in pGEM-MBLD plasmid,which conains MBL cDNA with codon 54 mutant allele,was amplified by PCR. After thecDNA fragement and plasmids pCI-neo were prepared by digestion with Sma I and Sal I,the fragment was inserted into Sma I and Sal I site in pCI-neo eukaryotic expression vector,and the recombinant vector,named pCI-MBL54,was obtained. The pCI-MBL54,digested with restriction enzymes,was found to contain the point mutation MBL cDNA by agarose gel electrophoresis analysis. The plasmids pCI-MBL54 containing full length of mutations MBL cDNA were propagated hi Escherichia coli XL-1 blue,then the extracted and purified pCI-MBL54 were used to transfect dhfr(-) Chinese-hamster ovary (CHO) cells. After screeened with G418 and cloned,4 G418-resistent clones were randomly selected for detection of mRNA expression by RT-PCR and molecular beacons. It was found that all of the 4 positive cell clones expresse MBL analogue as detected in transcription level.To prepare the wild type MBL in prokaryotic system,a pair of primers was designed and synthesized,and was used to amplify MBL gene from the recombinant vector pGEM-MBL that contans wild type MBL cDNA. A recombinant prokaryotic expression vector,pET28-MBL,was constructed by inserted the MBL gene into plasmid pET28(b),and after transfected it into Ecoli BL21 (DE3) and induced with IPTG,recombinant MBL protein was expressed successfully. SDS-PAGE and western-blot assay show that the expressed product was 29kDa and could be recognized by anti-6His McAb. The purified product was obtained by affinity chromatography and dialysis in 0.9%NaCl.As this work is a part of the project of mechanisms,diagnosis and therapy of MBL deficiency,it provides the basis for further research of MBL deficiency.