微生物学免疫学进展
微生物學免疫學進展
미생물학면역학진전
PROGRESS IN MICROBIOLOGY AND IMMUNOLOGY
2013年
4期
25-30
,共6页
赵雅静%魏然%冯德杰%刘晨鸣%魏至栋%高雪军%朱莉萍
趙雅靜%魏然%馮德傑%劉晨鳴%魏至棟%高雪軍%硃莉萍
조아정%위연%풍덕걸%류신명%위지동%고설군%주리평
轮状病毒( RV)%LLR株%NSP4基因%基因稳定性%序列分析
輪狀病毒( RV)%LLR株%NSP4基因%基因穩定性%序列分析
륜상병독( RV)%LLR주%NSP4기인%기인은정성%서렬분석
Rotavirus ( RV )%LLR strain%NSP4 gene%Gene stability%Sequencing
目的研究轮状病毒（ RV） LLR疫苗株NSP4基因的遗传特征及基因稳定性，为疫苗的质量控制提供依据。方法将LLR株轮状病毒毒种LLR38代在原代牛肾细胞上连续传至49代，提取第38、43、44、49代病毒RNA。通过RT-PCR扩增NSP4基因片段，将其克隆入质粒pGEM-T中，进行序列测定与分析。结果 LLR株NSP4基因全长751 bp，含编码175个氨基酸的单一的开放读码框架（ ORF）。各代次病毒的NSP4基因核苷酸与推导的氨基酸序列完全一致，与GenBank中LLR参考株（ AY219873）进行比较，核苷酸与推导的氨基酸同源性分别为99．1％和99．7％，各关键功能区均未发生改变。与27株A～F不同基因群RV代表株进行比较， LLR株与A基因群RV的NSP4同源性较高，核苷酸推导的氨基酸同源性为89．2％～95．5％；与B、C、D、E、F基因群RV核苷酸推导的氨基酸同源性分别为83．5％～87．5％、85．2％～87．5％、65．9％～67．8％、27．6％～30．8％、77．8％；系统发生树显示LLR株与A基因群RV在一个分支内。结论轮状病毒LLR疫苗株在原代牛肾细胞上连续传代NSP4基因遗传特性极其稳定，从分子水平证明LLR疫苗株毒种及其生产的疫苗是安全的。
目的研究輪狀病毒（ RV） LLR疫苗株NSP4基因的遺傳特徵及基因穩定性，為疫苗的質量控製提供依據。方法將LLR株輪狀病毒毒種LLR38代在原代牛腎細胞上連續傳至49代，提取第38、43、44、49代病毒RNA。通過RT-PCR擴增NSP4基因片段，將其剋隆入質粒pGEM-T中，進行序列測定與分析。結果 LLR株NSP4基因全長751 bp，含編碼175箇氨基痠的單一的開放讀碼框架（ ORF）。各代次病毒的NSP4基因覈苷痠與推導的氨基痠序列完全一緻，與GenBank中LLR參攷株（ AY219873）進行比較，覈苷痠與推導的氨基痠同源性分彆為99．1％和99．7％，各關鍵功能區均未髮生改變。與27株A～F不同基因群RV代錶株進行比較， LLR株與A基因群RV的NSP4同源性較高，覈苷痠推導的氨基痠同源性為89．2％～95．5％；與B、C、D、E、F基因群RV覈苷痠推導的氨基痠同源性分彆為83．5％～87．5％、85．2％～87．5％、65．9％～67．8％、27．6％～30．8％、77．8％；繫統髮生樹顯示LLR株與A基因群RV在一箇分支內。結論輪狀病毒LLR疫苗株在原代牛腎細胞上連續傳代NSP4基因遺傳特性極其穩定，從分子水平證明LLR疫苗株毒種及其生產的疫苗是安全的。
목적연구륜상병독（ RV） LLR역묘주NSP4기인적유전특정급기인은정성，위역묘적질량공제제공의거。방법장LLR주륜상병독독충LLR38대재원대우신세포상련속전지49대，제취제38、43、44、49대병독RNA。통과RT-PCR확증NSP4기인편단，장기극륭입질립pGEM-T중，진행서렬측정여분석。결과 LLR주NSP4기인전장751 bp，함편마175개안기산적단일적개방독마광가（ ORF）。각대차병독적NSP4기인핵감산여추도적안기산서렬완전일치，여GenBank중LLR삼고주（ AY219873）진행비교，핵감산여추도적안기산동원성분별위99．1％화99．7％，각관건공능구균미발생개변。여27주A～F불동기인군RV대표주진행비교， LLR주여A기인군RV적NSP4동원성교고，핵감산추도적안기산동원성위89．2％～95．5％；여B、C、D、E、F기인군RV핵감산추도적안기산동원성분별위83．5％～87．5％、85．2％～87．5％、65．9％～67．8％、27．6％～30．8％、77．8％；계통발생수현시LLR주여A기인군RV재일개분지내。결론륜상병독LLR역묘주재원대우신세포상련속전대NSP4기인유전특성겁기은정，종분자수평증명LLR역묘주독충급기생산적역묘시안전적。
Objective To study heredity characteristics and stability of NSP 4 gene of rotavirus ( RV) vaccine strain LLR and provide a basis for the quality control of vaccine .Methods Rotavirus strain LLR was subcultured in primary bovine kidney cells from passage 38 (LLR38) to passage 49, and the viral RNAs of passages 38, 43, 44 and 49 were extracted for amplification of NSP4 gene by RT-PCR.The amplified gene fragment were cloned into plasmid pGEM-T for sequencing. Resulst The NSP4 gene of LLR strain, with a full-length of 751bp, contained a single open reading frame (ORF) enco-ding 175 amino acids .The changes of nucleotides and deduced amino acids of strain LLR of various passages were com -pletely in agreement.The homologies of nucleotide and deduced amino acid sequences of strain LLR were 99.1% and 99.7%comparing with the reference strain LLR ( AY219873 ) in GenBank, and no significant change was observed in functional domains .Compared with the other 27 representative RV strains of NSP 4 genetypes A-F, the homologies of amino acid of strain LLR were 89.2%-95.5%to those of representative RV strain of genetypes A , while only 83.5%-87.5%, 85.2%-87.5%,65.9%-67.8%,27.6%-30.8%and 77.8%to those of representative RV strain of genetypes B ,C,D, E and F,respectively .LLR strain belongs to the same branch with genotype A on phylogenetic tree. Co nclusions The NSP4 gene of RV vaccine strain LLR showed highly genetic stability after continuously subcultured in primary bovine kidney cells.It proved a high level of safety of the LLR vaccine strain and prepared vaccine at molecular level .
