[Construction of eukaryote expression vector carrying human pulmonary surfactant-associated protein C and its expression in vitro].
OBJECTIVE
To clone human pulmonary surfactant-associated protein C (SP-C) and construct an eukaryote expression vector pcDNA3.1(+)/SP-C, and then to examine the SP-C expression in vitro, which may provide a reliable way of massive production of SP-C.


METHODS
The total RNA of normal lung tissue neighboring lung cancer from patients undergoing operation was extracted, and SP-C cDNA was then obtained by reverse transcription-polymerase chain reaction (RT-PCR). Both SP-C cDNA sequence and plasmid pcDNA3.1(+) were digested with NotI and XhoI, then connected by T4 DNA ligase after recycling agarose. After identification by restriction analysis and DNA sequencing, the recombinant was transfected into human breast cancer MCF-7 cells by using lipofectin reagent, and then the expression of SP-C was examined by RT-PCR and Western blotting.


RESULTS
Human SP-C cDNA could be correctly cloned into the plasmid pcDNA3.1(+), and SP-C protein may be expressed in MCF-7 cells after transfection.


CONCLUSIONS
By in vitro recombination, the eukaryote expression vector pcDNA3.1(+)/SP-C was successfully constructed and expressed SP-C in vitro, which rendered preparation for the construction of specific expression vector of human SP-C, and it laid the foundation of massive production of SP-C through mammary gland bioreactor.
Cui
Xiao-guang
XG
Department of Anesthesiology, Second Hospital Affiliated to Harbin Medical University, Key Laboratory of Anesthesiology and Critical Care Medicine of Heilongjiang Province, Harbin 150086, Heilongjiang, China.
Tan
Jing
J
Li
Cheng-ru
CR
Li
Wen-zhi
WZ
D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D037721:Pulmonary Surfactant-Associated Protein C / Q000235:genetics*
D037721:Pulmonary Surfactant-Associated Protein C