[Cloning and expression of full-length subtype E and subtype B gag gene from uncultured PBMCs of HIV-1 infected individuals in China].
OBJECTIVE
To clone and express subtype E and subtype B gag gene of the prevalent HIV-1 strain in China.


METHODS
One HIV-1 positive sample, whose subtype had been determined as sub-type E and one HIV-1 positive sample, whose subtype had been determined as subtype B by sequence analysis of partial env gene were chosen to be amplified by nested PCR. We got two full-length fragments of gag gene and incorporated them first into plin8Pr55 and then into pFastBacl.


RESULTS
Sequence analysis showed the cloned gag genes have the complete open-reading frames and have no major deletion and insertion. Using the Bac-toBac system, we obtained the recombinant baculoviruses containing gag genes. Western-blot analysis showed that insect cells infected with gag recombinant baculoviruses expressed HIV-1 gag antigen. Thin section electron microscopy showed that virus-like particles (VLPs) formed in insect cells was infected with gag.


CONCLUSIONS
The cloning and expression of gag genes of the prevalent HIV-1 stains in China revealed that the cloned genes have intact structure and function and this paves the way for the development of HIV-1 vaccines targeted at the epidemic of HIV-1 in China, also for the study of biological functions of gag gene and development of diagnostic kits of HIV-1.
Zhang
H
H
AIDS Reference Lab Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610081, China.
Shao
Y
Y
Xiao
Y
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D002681:China; D003001:Cloning, Molecular; D015870:Gene Expression; D015750:Genes, gag / Q000235:genetics*; D005822:Genetic Vectors; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D006801:Humans; D007313:Insecta / Q000166:cytology; D007963:Leukocytes, Mononuclear / Q000821:virology*; D017422:Sequence Analysis, DNA
D015699:HIV Envelope Protein gp120