Cloning of a fusion gene containing hGM-CSF gene and gene encoding L protein of HBV and expression in Pichia Pastoris.
OBJECTIVE
To construct the yeast expression vector containing cDNA sequence coding L protein of Hepatitis B Virus (HBV) and human granulocyte-macrophage colony stimulating factor (hGM-CSF), and to explore the method of secretory expression in Pichia Pastoris GS115 strain.


METHODS
We used pVAX1-L-hGM as template to amplify L-hGM gene by PCR, and then the PCR products were cloned into the yeast expression vector pPICZaC with DNA recombination method. After linearized by Sac I, the recombinant plasmid pPICZa C-L-hGM was transformed into Pichia Pastoris GS115 by electrophoresis. The expressing protein was induced by methanol. SDS-PAGE and Western blot were used to analyze the expression of protein.


RESULTS
DNA sequence analysis revealed that the constructed genes sequences were totally consistent with the GeneBank reported. The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in Pichia Pastoris.


CONCLUSIONS
The successful construction of a recombinant yeast expression vector containing gene coding L protein of Hepatitis B virus and hGM-CSF gene, and expressed in Pichia Pastoris, of which lays a foundation for the further researches on a better protective HB vaccine.
Li
Jun-Wu
JW
Microbiology and Immunology Department, Medical College, Jinan University, Guangzhou, China. ljw6610@163.com
Liu
Yan
Y
Li
Ke
K
Song
Dong
D
Ye
Qiu-Ping
QP
D015153:Blotting, Western; D003001:Cloning, Molecular; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics*; D006801:Humans; D010843:Pichia / Q000235:genetics*; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D014170:Transformation, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*
C096362:L protein, hepatitis B virus; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor