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以G1株系的甜菜坏死黄脉病毒(BNYVV)RNA3为模板,以Oligo(dT)_(15)为引物合成cDNA第一链,以RNA3特异引物合成cDNA第二链。然后将双链DNA克隆在pGEM3zf(+)载体中。通过限制性内切酶图谱分析,构建了五个亚克隆,完成全序列1776bp的序列测定(不含Poly(A)尾巴)。对核苷酸序列和推测的氨基酸序列分析结果发现,RNA3含三个开放阅读框架(ORF),分别编码三个多肽,即P25、P4.6和蛋白N。与法国F2株系RNA3在核昔酸水平上的同源率达96.8％,相应的CRF F25、ORF P4.6和ORFN编码多肽的同源率分别为93.6％、93.2％和92.3％。P25含一个钉指结构(zinc finger),和酸性及碱性区域;蛋白N为疏水蛋白。ORF以外的两端序列高度保守,且3’端可形成双发卡结构(double hairpin motif),说明这段序列在病毒侵染或复制中有十分重要的作用。进一步将RNA3 eDNA体外定点突变后使P25 ORS 与卢-半乳糖苷酶基因位于同一阅读框架以表达卢-半乳糖苷酶和P25融合蛋白,在诱导产物中检测到卢-半乳糖苷酶与P25的融合蛋白已在大肠杆菌中得到表达。
The first strand of cDNA was synthesized by using the cDNA of BNYVV RNA3 of G1 strain as the template and Oligo (dT) _ (15) as the primer, and the second strand of cDNA was synthesized by RNA3 specific primer. The double stranded DNA was then cloned into the pGEM3zf (+) vector. Five subclones were constructed by restriction endonuclease mapping, and the full-length 1776 bp sequencing (excluding Poly (A) tail) was completed. The nucleotide sequence and deduced amino acid sequence analysis revealed that RNA3 contains three open reading frames (ORFs), encoding three polypeptides, P25, P4.6 and N, respectively. The homology of nucleotide sequence of RNA3 to French F2 strain was 96.8%. The homologies of corresponding CRF F25, ORF P4.6 and ORFN-encoding polypeptide were 93.6%, 93.2% and 92.3%, respectively. P25 contains a zinc finger, and acidic and basic regions; protein N is a hydrophobin. Both ends of the ORF sequences are highly conserved, and the 3 ’end forms a double hairpin motif, indicating that this sequence plays a very important role in virus infection or replication. After further site-directed mutagenesis of RNA3 cDNA in vitro, the P25 ORS and the L-galactosidase gene were located in the same reading frame to express the L-galactosidase and P25 fusion proteins. The induction of L-galactosidase and P25 The fusion protein has been expressed in E. coli.