THE EXPRESSION OF CHLORAMPHENICOL ACETYLTRANSFERASE IN AEDES-ALBOPICTUS (C6/36) CELLS AND AEDES-TRISERIATUS MOSQUITOS USING A DOUBLE SUBGENOMIC RECOMBINANT SINDBIS VIRUS

被引:36
作者
OLSON, KE
HIGGS, S
HAHN, CS
RICE, CM
CARLSON, JO
BEATY, BJ
机构
[1] UNIV VIRGINIA,HLTH SCI CTR,DEPT MICROBIOL,CHARLOTTESVILLE,VA 22908
[2] UNIV VIRGINIA,HLTH SCI CTR,BEIRNE CARTER CTR IMMUNOL RES,CHARLOTTESVILLE,VA 22908
[3] WASHINGTON UNIV,SCH MED,DEPT MOLEC MICROBIOL,ST LOUIS,MO 63110
关键词
SINDBIS VIRUS EXPRESSION SYSTEM; CHLORAMPHENICOL ACETYLTRANSFERASE; AEDES-TRISERIATUS; AEDES-ALBOPICTUS CELLS;
D O I
10.1016/0965-1748(94)90121-X
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/CAT virus was used to infect C6/36 (Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4.0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCID50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypeptides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(11) CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.
引用
收藏
页码:39 / 48
页数:10
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