Construction of armored RNA containing long-size chimeric RNA by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the MS2 bacteriophage.
OBJECTIVE
To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA by increasing the number and affinity of the pac site.


METHODS
The plasmid pET-MS2-pac was constructed with one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified.


RESULTS
The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions.


CONCLUSIONS
The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences.
Wei
Baojun
B
Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, National Center for Clinical Laboratory, Beijing Hospital, Beijing, PR China.
Wei
Yuxiang
Y
Zhang
Kuo
K
Yang
Changmei
C
Wang
Jing
J
Xu
Ruihuan
R
Zhan
Sien
S
Lin
Guigao
G
Wang
Wei
W
Liu
Min
M
Wang
Lunan
L
Zhang
Rui
R
Li
Jinming
J
D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017909:Levivirus / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000378:metabolism; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D036022:Capsid Proteins; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase; D012260:Ribonucleases