新城疫病毒V4株F基因的克隆与核苷酸序列分析

Cloning and Sequencing of F Gene of Newcastle Disease Virus V4(Hr) Strain

本试验采用RT_PCR方法对NDVV4 克隆株V4(Hr)的F基因进行了扩增与克隆,并以Sanger’s 双脱氧末端终止法测定出其核苷酸序列,推导出氨基酸序列。F基因全长为1662bp,单一的开放阅读框编码553 个氨基酸的长肽。F蛋白的疏水构型有3 个强疏水区;F蛋白上共有12 个Cys 残基位点和5 个潜在糖基化位点。同其亲本毒株Queensland 相比,核苷酸同源率为99.3% ,氨基酸同源率98.7% 。上述主要功能区的序列完全一致,但其中7 个氨基酸的不同,的确导致了二级结构的变化(主要表现为α_螺旋、β_折叠、β_转角和β_卷曲的数量和位置的不同),而且单一的氨基酸差异足以导致二级结构的变化,如:214位的P对L的替换,就导致212 ～215

Fusion glycoprotein (F) gene of NDV V4(Hr) strain was amplified by reverse transcriptase_polymerase chain reaction(RT_PCR)and cloned into the pGEM_T vector. The sequence of the cDNA was obtained by Sanger's sequencing technique with their amino acid sequence being deduced.The results indicated that F gene is 1662bp in length and has a single opening read frame, which code for a polypeptide of 553 amino acid. F glycoprotein has three highly hydrophobic domains. There are 12 Cysteine residues and 5 potential asparagine_linked glycosylation sites in F glycoprotein. Comparison of the nucleotide sequence and deduced amino acid scquence with that of its' parent strain Queensland showed that a high degree of functional and structural constraint exist on the F glycoprotein. F gene nucleotide acid sequence exhibited homology of 99.3% and amino acid 98.7%.However,7 amino acid residues variations in coding region of F gene did cause the appear of secondary structural differences between V4(Hr)and Queensland strain of NDV,and exhibited as the difference of number and positions of α_helix, β_folding,β_turn and β_coil.Even if one residues (such as P at site 214 to L)can cause the secondary structure change. Fusion glycoprotein (F) gene of NDV V4(Hr) strain was amplified by reverse transcriptase_polymerase chain reaction(RT_PCR)and cloned into the pGEM_T vector. The sequence of the cDNA was obtained by Sanger's sequencing technique with their amino acid sequence being deduced.The results indicated that F gene is 1662bp in length and has a single opening read frame, which code for a polypeptide of 553 amino acid. F glycoprotein has three highly hydrophobic domains. There are 12 Cysteine residues and 5 potential asparagine_linked glycosylation sites in F glycoprotein. Comparison of the nucleotide sequence and deduced amino acid scquence with that of its' parent strain Queensland showed that a high degree of functional and structural constraint exist on the F glycoprotein. F gene nucleotide acid sequence exhibited homology of 99.3% and amino acid 98.7%.However,7 amino acid residues variations in coding region of F gene did cause the appear of secondary structural differences between V4(Hr)and Queensland strain of NDV,and exhibited as the difference of number and positions of α_helix, β_folding,β_turn and β_coil.Even if one residues (such as P at site 214 to L)can cause the secondary structure change.