[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.
Liu
Feng-ling
FL
Department of Immunology, Southern Medical University, Guangzhou 510515, China.
Zuo
Da-ming
DM
Bai
Zhi-jun
ZJ
Chen
Zheng-liang
ZL
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