[Construction of the expression vector PcDNA4/His C-MBL and its expression in Chinese-hamster ovary cells].
OBJECTIVE
To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells.


METHODS
The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay  ELISA  using the anti-serum from Balb/C mice immunized with the recombinant protein.


RESULTS
The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA.


CONCLUSIONS
The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.
Department of Immunology, First Military Medical University, Guangzhou 510515, China.
Zhi-jun
ZJ
Bai
Li-yun
LY
Zhang
Zuo-xian
ZX
Lin
Xiao
X
Lu
Zheng-liang
ZL
Chen
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics*; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000235:genetics; D018274:Electroporation; D005822:Genetic Vectors; D037601:Mannose-Binding Lectin / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D018076:DNA, Complementary; D004274:DNA, Recombinant; D037601:Mannose-Binding Lectin; D012333:RNA, Messenger; D011994:Recombinant Proteins