马传染性贫血病毒部分致弱毒株体外和体内的进化分析

【作者】 李强；

【导师】 韩德权； 周建华；

【作者基本信息】 黑龙江大学 ， 微生物学， 2009， 硕士

【摘要】 我国研制成功的马传染性贫血病毒驴白细胞弱毒疫苗株DLV是将一株来源于马体的EIAV强毒LV (辽毒株)经过驴体连续传代100余代使其病毒毒力增强后获得了驴强毒株DV(对马和驴致死率为90%),然后将该驴强毒株通过驴白细胞体外连续培养驯化120余代,使该毒株病毒毒力丧失的同时又保持了良好的免疫原性,最终培育成功了EIAV驴白细胞弱毒疫苗。将驴白细胞弱毒疫苗株进一步通过驴胎皮肤细胞继代,又获得比驴白细胞弱毒疫苗免疫保护效果更好的驴胎皮肤细胞弱毒疫苗FDDV。为探讨驴胎皮肤细胞弱毒疫苗株FDDV在免疫成熟马体内的进化规律,本研究采用nest PCR方法对FDDV免疫成熟马(FDDV免疫24个月后)体内的低拷贝的FDDV前病毒基因组DNA进行了分段(5段)扩增和序列分析,旨在找出FDDV长期在免疫马体内的进化规律并推导出其在体内的优势序列,通过与体外FDDV基因组进行比较分析,找到驴胎皮肤细胞弱毒疫苗株FDDV体内体外演化过程中保守及变化区域。最终每段得到5个阳性克隆的序列。通过比较分析发现:免疫马体内LTR与FDDV3-8 LTR的同源率平均为98.8%,env编码的氨基酸序列的同源率平均为96.1%。免疫马体内外FDDV的囊膜蛋白gp90变异较大,与体外gp90相比免疫马体内gp90的N-糖基化位点有增加现象。为阐明EIAV疫苗的致弱机制和免疫保护机理,本研究采用LA-PCR技术对驴白细胞弱毒疫苗株减毒过程中第92代次毒株(DLV92)前病毒基因组DNA进行了扩增和序列分析,旨找出潜在的与毒力变化和免疫保护相关的稳定变异位点和相关特性。本实验成功的得到了8株接近DLV92全长的8Kb的前病毒基因组核酸序列。通过比较分析得出:DLV92与DLV878(疫苗株DLV ,GenBank中登陆号为AF327878)同源率平均为98.1%,与LV877 (EIAV强毒辽宁株LV,GenBank中登陆号为AF327877)核苷酸序列的同源率平均为97.7%。DLV92囊膜蛋白gp90上存在17个N-糖基化位点,与疫苗株DLV878一致,比亲本强毒LV877的囊膜蛋白gp90少4个糖基化位点,比DLV61少2个糖基化位点。本研究结果将为进一步研究EIAV致弱过程中毒力的变化和免疫保护的分子机制奠定基础。更多还原

【Abstract】 The equine infectious anemia virus donkey leukocyte attenuated vaccine DLV had been used to protect against equine infectious anemia (EIA) disease for several decades in China. The vaccine had good immunogical protection and safety, providing a unique natural model system for other retroviruses including HIV. The generation of Chinese EIAV attenuated vaccine included three stages. Firstly, a carefully selected field isolate was passaged in horses for 16 generations to obtain a pathogenic strain, named EIAVL strain. Secondly, VL strain was then passaged 100 times in donkeys resulting to gain a more virulent strain EIAVD strain which yielded 100% acute EIA symptom and more than 90% mortality in experimentally infected horses in the first 50 days after inoculation; finally, the DLV vaccine was developed from EIAVD strain through 120 successive passages in vitro in primary donkey macrophages. Thereafter, by the same method, another fetal donkey dermal cells adapted vaccine was developed by passaging EIAVDLv in fetal donkey dermal FDDV cells for 15 times and FDDVvaccine was proved to have more excellent effect of immune protection than that of EIAVDLV vaccine.To discuss the FDDV genome evolution in vivo in inoculated horses, the provirus DNA of the EIAV in the FDDV vaccinated experimental horse which had been inoculated for 24 mouths were amplified by nest PCR. Based on the analysis about the obtained FDDV provirus DNA sequences, we hope to identify the FDDV genome evolution character in vivo in horses after a long time immunization, those included which regions in FDDV genome were conserved and which ones were variable. Our results showed that: Compared with the standard EIAVFDDV genome sequence come from a FDDV molecular clones FDDV3-8,the amino acids sequences predicted based on obtained DNA sequences in vivo showed a 98.8% and 96.1% homologous on average in LTR and ENV regions. We found that more variations occur in env region of the EIAVFDDV genome, and the number of N-glycoslation sites in env of the FDDV increased after a 24 months replication in vivo.To elucidate the mechanism of EIAV vaccine on the attenuated virulence and enhanced immunogenecity, the proviral genomic DNA of a DLV strain isolated at the 92th generation of attenuation was cloned by LA-PCR and analyzed. We obtained eight full genomic clones of DLV92 randomly in this experiment, and each individual gene of the genome was compared with the corresponding sequences of Chinese EIAV virulent strain LV (Genebank Acc.#: AF327877)and the vaccine strain DLV(Genebank Acc. #:AF327877. The compared results showed that the nucleotide homologous on average between DLV92 and DLV878 was 98.1%, and 97.7% homologous on average was showed between DLV92 and LV.17 N-glycoslation sites were discovered in membrane protein gp90 genes of the DLV92. This is consensus with the N-glycoslation sites in EIAV vaccine strain DLV878, but 4 less than LV877 and 2 less than DLV61. Results presented in this article provided valuable information for the intensive study of the mechanism on attenuation and inducing protective immunity of Chinese EIAV vaccine.更多还原