Abstract:

BACKGROUND: Studies have suggested that gene defection leads to the structural change of pulmonary surfactant protein C (SP-C). Early detection of pulmonary surfactants is of great significance for prediction of occurrence of lung diseases.
OBJECTIVE: To clone human pulmonary SP-C, to construct an prokaryotic expression vector PET28a/SP-C, and to obtain purified SP-C.
METHODS: The total RNA of normal lung tissue was extracted, and SP-C cDNA was then obtained by reverse transcription-polymerase chain reaction. The purified product of SP-C cDNA was then inserted into PMD-18T vector to obtain recombinant PMD-18T/SP-C. The recombinant plasmid PMD-18T/SP-C was cut by restriction enzymes Bam HⅠ/Hind Ⅲ and then purified to become SP-C cDNA with viscous ends, as was PET28a to become a linear plasmid fragment with the same viscous ends as SP-C cDNA. The SP-C cDNA was combined with the PET-28a that had been cut by the enzymes to construct the recombinant plasmid PET-28a/SP-C. The correct PET-28a/SP-C was transformed into BL21 to induce expression.
RESULTS AND CONCLUSION: Two bands were detected at 5 000-7 500 bp and 250-1 000 bp after SP-C cDNA and its recombinant plasmid were identified after Bam HⅠ and Hind Ⅲ double digestion. The fragment length of the inserted gene was 597 bp, in consistence with the SP-C cDNA sequence announced by GeneBank. Western-blot analysis showed that the purified SP-C protein had a new band with the expected size at a relative molecular mass of about 27 000. These findings indicate that human SP-C cDNA can be correctly cloned into the PET-28a, with construction of the recombinant plasmid PET-28a/SP-C, and SP-C protein may be expressed in BL21.