Construction and expression of ribonuclease-resistant virus-like particles containing long chimeric RNA sequence / 中华检验医学杂志

ABSTRACT

Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop.Methods 1.7 kb maturase and coat protein gene of ms2 were amplified.We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine.The 1700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac.The five target DNA sequences were spliced by overlapping extension(including 3 fragment of SARS-CoV,one fragment of HCV and one fragment of HSN1).PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM(R) T-easy vector and then excised with the Not Ⅰ restriction enzymes from the resulting recombinant plasmid.simoutaniously the pET-MC-pac plasmid was digested with Not Ⅰ restriction enzymes,fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V.Simoutaniously,we constructed three recombinant expression vector for control,including N-P3V-pET-P、N-P3V-pET-Cand P-3V-pET-P.N-P3V-pET-P contained one wild type pacsite located behind ms2 sequenceN-P3V-pET-C contained one C-variant pacsite located behind ms2 sequenceP-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence,another is located between SARS-CoV 3 and HCV sequence.Then these four expression vector were transformed into E.coli strain BL21(DE3),respectively.The 3V Armored RNA was expressed and purified.A260 absorbance value of expression product was determined.Results Four expression plasmids were constructed successfully.The Armored RNA with 1891 bases was snccessfully expressed by the two-paesite expression system of pET-ms2-3V and P-3V-pET-P;for N-P3V-pET-P、N-P3V-pET-C,only 1 200 bp target sequence was packaged into virus-like particles.Expression efficiency of N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P and N-P3V-pET-P was 0.23,0.35,0.35,0.51 mg/ml,respectively.Armored RNA was shown to have the charaeterization of Ribonuclease-resistant and stable at 4℃,37℃ and 25℃ respectively.Conclusion The expression plasmids containing two pacsites were constructed successfully and prokaryotic expression system can be used as an expressing plasmid platform for preparation of Ribonuclease-resistant virus-like particles containing long chimeric RNA sequence.