[Construction of codon-optimized HPV16 capsid genes in the eukaryotic co-expression vector and transfection of cells].
OBJECTIVE
To construct the eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 for harvesting sufficient amounts of infectious HPV16.


METHODS
The amplified codon-optimized HPV16 capsid genes from 988 plasmid by PCR were cloned into pCR-XL-TOPO vector and then subcloned into eukaryotic expressing vector pcDNA3.1(+). Thus, eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 capable of expressing HPV L1 gene and L2 gene was constructed. This eukaryotic vector was verified by enzymolysis and sequencing. The transcription of capsid genes was observed in vivo and in vitro by hydrodynamics-based transfection and liposome-mediated transfection. The expression of L1 gene in 293T cells was examined by Western blot.


RESULTS
The eukaryotic co-expression vector pcDNA3.1-L1-IRES-L2 was successfully constructed and verified by enzymolysis and sequencing.The recombinant plasmid L1 and L2 genes were transcripted in the livers of the rats and 293T cells.Cytopathic effect (CPE) occurred after the transfected with pcDNA3.1-L1-IRES-L2 into 293T cells, indicating that the two capsid genes were expressed.Western blot detection showed the recombinant plasmid L1 protein was expressed in 293T cells.


CONCLUSIONS
The pcDNA3h1-L1-IRES-L2 has been constructed successfully, which lays a foundation for in-dept study on the infectious mechanism of HPV16.
Liu
Li-Hong
LH
Key Laboratory of Molecular Biology, College of Life Science and Technology, Xinjiang University, Key Laboratory of Xinfiang Biological Resources and Genetic Engineering, Urumqi 830046, China.
Xu
Lu
L
Li
Yi-Jie
YJ
Zhang
Fu-Chun
FC
Ma
Zheng-Hai
ZH
D000818:Animals; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003062:Codon / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008081:Liposomes / Q000737:chemistry; D051379:Mice; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D036022:Capsid Proteins; D003062:Codon; D008081:Liposomes; D011993:Recombinant Fusion Proteins