[Construction of standard recombinant plasmids for 7 common mannan-binding lectin gene haplotypes].

OBJECTIVE

To construct the standard recombinant plasmids for 7 common haplotypes of mannan-binding lectin (MBL) gene.

METHODS

The DNA samples with known haplotypes and genotypes of MBL gene were used as the templates for amplifying the fragments of MBL gene haplotypes including the promoter region and exon 1 with sequence-specific primer-polymerase chain reaction (SSP-PCR) method. The amplified fragments were cloned into T vector and the bases located at codon 52 and codon 57 of exon 1 in MBL gene were mutated respectively by site-directed mutagenesis. All the 7 recombinant plasmids were identified by PCR and direct sequence analysis.

RESULTS

From the DNA samples with known haplotypes and genotypes of MBL gene, the standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA and LYPB of MBL gene were constructed by SSP-PCR and molecular cloning technique. From the recombinant plasmids of HYPA and LYQA, the standard plasmids of haplotypes HYPD and LYQC of MBL gene were constructed by site-directed mutagenesis, respectively.

CONCLUSION

The constructed standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene provide standard controls for detecting the SNPs, haplotypes and genotypes of MBL gene with such genotyping methods us SSP-PCR and real-time PCR.