[Expression of human CGT52TGT MBL mutant in CHO cells and analysis of the expression product].
OBJECTIVE
To explore preliminarily the mechanisms of immunodeficiency resulted from the CGT52TGT point mutation of mannan-binding lectin(MBL) gene.


METHODS
The MBL gene containing CGT52TGT point mutation was amplified from the plasmid pMBLm52 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. After confirmed by DNA sequencing, the recombinant expression vector was transfected into Chinese-hamster ovary(CHO) cells by electroporation. Zeocin of 800 mg/L had been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain stable transfectant. The expression of mRNA was analyzed by RT-PCR, the recombinant protein was purified from the culture supernatant by Ni-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing condition and Western blot.


RESULTS
The cDNA fragment amplified from pMBLm52 plasmid by PCR was about 750 bp and the recombinant plasmid pcDNA4/HisMax C-MBLm52 was constructed and transfected into CHO cells. The expression product purified from the culture supernatant appeared mainly at the site of M(r) 60,000, indicating a much lower oligomerization level than that of the recombinant human wild MBL and human plasma-derived MBL.


CONCLUSIONS
The CGT52TGT point mutation of MBL gene does not affect the secretion of its product, but a Cys introduced by the mutation could form another disulfide bond which may disrupt the structure of MBL molecule as well as its function.
Department of Immunology, Southern Medical University, Guangzhou 510515, China.
Feng-ling
FL
Liu
Da-ming
DM
Zuo
Zhi-jun
ZJ
Bai
Zheng-liang
ZL
Chen
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D006801:Humans; D037601:Mannose-Binding Lectin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D037601:Mannose-Binding Lectin; D050505:Mutant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins