[Expression of anti-HIV envelope glycoprotein scFv in Pichia pastoris and its biologic activities analysis].
OBJECTIVE
To construct the vector and express anti-HIV-1 envelope glycoprotein single chain Fv fragment in Pichia pastoris.


METHODS
The target gene was digested from plasmid pET28-scFv and cloned into pichia pastoris vector via gene engineering and DNA recombination techniques. The recombinant plasmid was linearized and transferred into Pichia pastoris strains GS115 by electroporation. After positive recombinant was selected and expression was induced by methanol, the target protein was analyzed by RT-PCR, SDS-PAGE and double-antibody sandwich ELISA.


RESULTS
High copies of transformant were obtained by phenotype determining and PCR amplification. RT-PCR and SDS-PAGE demonstrated the target protein was successfully expressed. And the yield account for about 18 percent of the total cell proteins. Double-antibody sandwich ELISA analysis proved that the recombinant scFv had good biological activities since it could be recognized and induce special immune respond with gp120 antigen.


CONCLUSIONS
The scFv was expressed successfully. This research will lay the foundation for AIDS target therapy and further study of anti-HIV activities.
Virus Laboratory of Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, China. lichang78@163.com
Chang
C
Li
Ning-Yi
NY
Jin
Hong-Wei
HW
Wang
Yu-Sheng
YS
Liu
Fang
F
Yu
Jing-Shan
JS
Tong
Ning-Ning
NN
Hu
D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D015497:HIV-1 / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054299:env Gene Products, Human Immunodeficiency Virus / Q000276:immunology*
D007135:Immunoglobulin Variable Region; D054299:env Gene Products, Human Immunodeficiency Virus