Olson K E, Higgs S, Hahn C S, Rice C M, Carlson J O, Beaty B J
Arthropod-borne and Infectious Disease Laboratories (AIDL), Colorado State University, Fort Collins 80523.
Insect Biochem Mol Biol. 1994 Jan;24(1):39-48. doi: 10.1016/0965-1748(94)90121-x.
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.
基因组RNA在体外从双亚基因组重组辛德毕斯（SIN）病毒表达载体pTE/3'2J/CAT转录而来，并转染到BHK-21细胞中以产生重组病毒储备液。TE/3'2J/CAT病毒用于感染C6/36（白纹伊蚊）细胞和成年雌性三带喙库蚊。当以大于20的感染复数（MOI）用TE/3'2J/CAT病毒感染C6/36细胞时，100%的细胞表达氯霉素乙酰转移酶（CAT）。感染后24小时（pi）每个细胞表达的CAT多肽数量为8.3×10⁵。将约4.0 log₁₀半数组织培养感染剂量（TCID₅₀）的TE/3'2J/CAT病毒经胸腔接种到成年雌性蚊子体内。接种后4天内检测到滴度大于6.0 log₁₀TCID₅₀/ml，接种后20天下降至小于4.0 log₁₀TCID₅₀/ml。接种后2天内检测到CAT活性（8×10⁻⁵单位的CAT/蚊子或1.4×10¹⁰个CAT多肽），在第6天达到峰值（4×10⁻³单位的CAT/蚊子或7.2×10¹¹个CAT多肽），并在第20天保持在峰值水平。免疫荧光和CAT活性测定用于在受感染蚊子中定位CAT表达，并证明CAT存在于神经、中肠、卵巢和唾液腺组织中。基于甲病毒的表达载体应可用于在蚊子细胞以及成年蚊子中表达异源基因。
