摘要
本研究对河北省保定地区鸡场分离到的两株鸡新城疫病毒(NDV)强毒株(代号分别为HBB和HBW),以及国内标准强毒株F_(48)E_9、疫苗株LaSota的F蛋白裂解位点附近的基因进行了RT-PCR扩增、克隆及序列测定。对克隆测序的4株鸡新城疫病毒的F基因与不同基因型的代表株进行同源性比较和毒力分析。并且对这4株新城疫病毒及Genbank上国内外有代表性的33株新城疫病毒共计37株F基因的389bp(1bp～389bp)长度的核苷酸片段及其相应的氨基酸序列(1aa～130aa)进行比较,建立系统发育进化树,并确定测序的4株新城疫病毒的基因型。结果显示,疫苗株LaSota同两分离株的同源性较低,核苷酸的同源性在83.8%～84.1%之间,氨基酸的同源性在84.4%～86.6%之间;国内标准强毒F_(48)E_9与LaSota及两分离株的同源性较低,核苷酸的同源性在85.6%～86.4%之间,氨基酸的同源性在84.4%～86.6%之间;两分离株核苷酸序列同源性较低,为88.2%,但氨基酸序列同源性较高,为93.9%。F_(48)E_9株F蛋白的裂解位点为~(112)RRQRRF~(117),两株强毒株F蛋白的裂解位点为~(112)RRQRRF~(117),符合强毒株裂解位点的特征,同毒力测定结果相一致。两分离株的F基因都没有碱基的缺失和插入,但有多处点突变,分散存在。分离株的55～61、72、84～89及128～130之间的氨基酸均高度保守,突变的氨基酸位点多集中在101～111之间。分离株HBB为基因Ⅵ型,分离株HBW为基因Ⅶ型。
同时,采用分离的基因Ⅶ型新城疫病毒毒株制成油乳剂灭活疫苗,分别以分离株和LaSota株的灭活苗免疫试验鸡,进行疫苗免疫效果比较和交叉免疫保护试验。结果显示,免疫组与对照组相比HI抗体效价差异显著,分离株灭活苗免疫组产生的HI抗体效价和LaSota灭活苗免疫组的抗体效价相差不明显,两组的升降规律也基本一致;分离株灭活苗免疫组(HI抗体效价为6log2以上时)在免疫后21、28和35天对相同剂量F_(48)E_9标准毒株和两分离株的攻毒均给予100%的保护,LaSota灭活苗免疫组(HI抗体效价为6log2以上时)对F_(48)E_9标准毒株也都给予100%的保护,对分离株保护率,除基因Ⅵ型HBB毒株在免疫后21和28天达100%外,其余仅为80%,表明NDV不同基因型毒株间的抗原性存在差异。
将遗传变异分析和交叉免疫保护试验的结果相结合,进行综合分析,结果显示疫苗株LaSota同现今流行毒株之间存在一定的抗原性差异。
The genes in the protein F cleavage site region of 4 strains of Newcastle disease virus (NDV) were cloned and their gene sequences were measured after amplified by RT-PCR. Two strong virulent strains are isolated from the NDV-infected poultry farms in Baoding of Hebei Province, and were respectively named HBB and HBW. Two strains of the others were the standard strong virulent strain F_(48)E_9 and the vaccinal strain LaSota. Then their homogeneity was compared with those of different genotype typical strains and their violence was analyzed. Gene F of 389 bp (1-389 bp) and their corresponding amino acid sequence(1-130 amino acid residues) of 37 NDV strains, including the 4 strains and 33 typical strains from Genbank, were compared and analyzed. The phylogenetic tree system was established, and the genotypes of the 2 isolated strains have been decided. The result showed that there is a low homogeneity between F_(48)E_9 and LaSota or the two isolated strong virulent strains, with homogeneity ranging from 83.8% to 84.1% in nucleotide and 84.4%~86.6% in amino acid residue. However, between two isolated strong virulent strains, there was a low homogeneity of 88.2% in nucleotide, and there was a high homogeneity of 93.9% in amino acid residue. The cleavage site of F protein of F_(48)E_9 is ~(112)RRQRRF~(117), while the two strains isolates have the cleavage site-~(112)RRQKRF117, which is in agreement with the characteristics of high virulent strains and the results of pathogenic tests. None of the F genes of the isolated NDV virulent strains suffered any base deletion and insertion, but there are mutations in many points scattering about. The amino acids of 55 to 61, 72, 84 to 89 and 128 to 130 in the isolated strains are all highly conservative, and their mutation points were mainly located between 101 and 111. The genotype of HBB in the isolated NDV strains belongs to the typical genotypeⅥ, and that of the HBW belongs to the genotype Ⅶ.At the same time, inactivated oil-emulsion vaccine was made by isolated strain of genotype Ⅶ of NDV. After the vaccination with the inactivated LaSota strain vaccine and inactivated isolated strain vaccine, their immuning effects were compared, and cross-protection test. The result showed that there is apparent difference in the antibody titer of HI between the samples from vaccination group and the control group. There is no apparent difference in the antibody titer of HI between chickens inoculated with isolates inactivated vaccine and inactivated LaSota vaccine during all experiment period. There is also a same varing tendency between the two groups. At the days of 21, 28 and 35 after inoculation (the HI titer were higher than 61og2) with isolated inactivated vaccine could
obtain 100% protection against the attacks both with F4gE9 and the isolated strains. LaSota inactivated vaccine (the HI titer were higher than 61og2) could provide 100% protection against the attack with F48E9 in these periods and the attacks with genotype VI of HBB strain. The protection rate against other isolated strains was 80%, having not reach 100%. It indicated there are differences in the antigenicity among the strains in different genotypes of NDV.Comprehensive analyses of the cross-protection and genetic variation showed that that the antigenic difference variation does exist among the vaccinate strain of LaSota and the currently epidemic strains of NDV.
引文
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