Construction Of Coxsackievirus B_3m Full Length CDNA Library And Screen Of Positive Clones

Objective: To investigate the molecular mechanism of child viral myocarditis caused by CVB3m, we constructed CVB3m cDNA library and screened positive clones and amplified 2B gene which encodes 2B protein. Methods: BalbIC newborn mice were infected with coxsackievirus B3m ,and mRNA was extracted from the myocardial tissue of CVB3m-induced myocarditis mice. The mRNA containing poly(A) was isolated by chromatography on oligo(dT) cellulose and reversely transcripted into the first-strand eDNA. The second- strand DNA was synthesized by using RNase H and DNA polymerase I, and E.coli ligase to replace the RNA strand with deoxynucleotides. After dephosphorylation and addition of EcoR I/Not I adaptor, the cDNA was inserted into EcoR I-digested lambda gtl 1 vector. The ligated DNA was packaged .The cDNA libray was screened using a complement , immunological method with anti-CVB3m mouse convalescent polyclonal sera ,and in situ hybridization of nucleicotide with DIG-labled CVB3m 2B gene probe. The positive clones were amplified by PCR with 2B primers and CVB3m primers . Results: CVB3m eDNA expression library was constructed , and the cloning capacity was 3.18 X 106 recombinants, the titration of packaged phage was up to 1.59X ~ and the inserted proportion accounted for 71%. Forty potential positive clones were retrieved by immune technique, and eight positive clones were obtained by in situ hybridization of nucleicotide. We succeeded in amplii~抜ng 2B gene by PCR. Conclusion: CVB3m cDNA expression library is constructed and the positive clones are screened, which lays a foundation for the explanation of toxicity of 2B protein of CVB3m and the further study of its pathological mechanism.