中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2008年
3期
280-286
,共7页
魏玉香%张括%魏葆珺%王露楠%张瑞%李金明
魏玉香%張括%魏葆珺%王露楠%張瑞%李金明
위옥향%장괄%위보군%왕로남%장서%리금명
轻病毒属%核糖核酸酶类%病毒粒子%病毒装配%病毒包膜蛋白质类
輕病毒屬%覈糖覈痠酶類%病毒粒子%病毒裝配%病毒包膜蛋白質類
경병독속%핵당핵산매류%병독입자%병독장배%병독포막단백질류
Levivirus%Ribonucleases%Virion%Virus assembly%Viral envelope proteins
目的 通过改变原噬菌体ms2包膜蛋白RNA包装位点(19碱基的茎环结构)的数量及亲和力,构建新的原核表达系统,探讨表达内含长片段(达到理论上的1 900 bp)RNA的耐RNase病毒样颗粒的可能性.方法 首先设计含Hind Ⅲ和Not Ⅰ酶切位点的引物,扩增ms2包膜蛋白的编码成熟酶蛋白和衣壳蛋白的1 700 bp序列,并将原来的19mer的包装位点序列改变为C-5变异体(19 bp stem-loop结构中-5位的尿嘧啶改变为胞嘧啶),Hind Ⅲ和Not Ⅰ酶切后,与用同样酶切的表达载体pET-28(b)相连接,得到重组载体pET-ms2-pac.应用重叠PCR扩增3种病毒的5段嵌合体序列(包括3段SARS-CoV基因、一段HCV基因和一段HSN1基因),并在SARS-CoV3和HCV序列之间插入一个19mer的变异体包装位点序列,在设计引物时,使嵌合体两端含有Not Ⅰ酶切位点,与Not Ⅰ酶切的重组载体pET-ms2-pac相连接,构建得到具有2个变异包装位点的表达载体pET-ms2-3V.同时构建3种对照重组表达载体,分别测定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V 4种重组表达质粒表达产物的260 nm吸光度(A260)值,根据公式A160=0.125 mg/ml计算4种表达产物的表达效率.结果 成功构建了4种原核表达载体:pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C.pET-ms2-3V和P-3V-pET-P经原核表达后得到含全长为1 891的5段嵌合体RNA的病毒样颗粒;N-P3V-pET-P、N-P3V-pET-C其原核表达产物病毒样颗粒中仅包装了1 200 bp的目的 嵌合体RNA.N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的表达效率分别为0.23、0.35、0.35和0.51 mg/ml.N-P3V-pET-C比N-P3V-pET-P表达效率高52%,而pET-ms2-3V比P-3V-pET-P表达效率高38%.所包装的RNA具有耐RNase和DNase消化的特性以及良好的不同温度条件下的稳定性.结论 通过改变噬菌体ms2 RNA包装位点(19碱基的茎环结构)的数量,可构建能表达内含达到理论上的约1 900 bp外源RNA的耐RNase病毒样颗粒的原核表达载体平台.通过应用包装位点的变异体C-变异体,可以增加病毒样颗粒的表达效率.
目的 通過改變原噬菌體ms2包膜蛋白RNA包裝位點(19堿基的莖環結構)的數量及親和力,構建新的原覈錶達繫統,探討錶達內含長片段(達到理論上的1 900 bp)RNA的耐RNase病毒樣顆粒的可能性.方法 首先設計含Hind Ⅲ和Not Ⅰ酶切位點的引物,擴增ms2包膜蛋白的編碼成熟酶蛋白和衣殼蛋白的1 700 bp序列,併將原來的19mer的包裝位點序列改變為C-5變異體(19 bp stem-loop結構中-5位的尿嘧啶改變為胞嘧啶),Hind Ⅲ和Not Ⅰ酶切後,與用同樣酶切的錶達載體pET-28(b)相連接,得到重組載體pET-ms2-pac.應用重疊PCR擴增3種病毒的5段嵌閤體序列(包括3段SARS-CoV基因、一段HCV基因和一段HSN1基因),併在SARS-CoV3和HCV序列之間插入一箇19mer的變異體包裝位點序列,在設計引物時,使嵌閤體兩耑含有Not Ⅰ酶切位點,與Not Ⅰ酶切的重組載體pET-ms2-pac相連接,構建得到具有2箇變異包裝位點的錶達載體pET-ms2-3V.同時構建3種對照重組錶達載體,分彆測定N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V 4種重組錶達質粒錶達產物的260 nm吸光度(A260)值,根據公式A160=0.125 mg/ml計算4種錶達產物的錶達效率.結果 成功構建瞭4種原覈錶達載體:pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P和N-P3V-pET-C.pET-ms2-3V和P-3V-pET-P經原覈錶達後得到含全長為1 891的5段嵌閤體RNA的病毒樣顆粒;N-P3V-pET-P、N-P3V-pET-C其原覈錶達產物病毒樣顆粒中僅包裝瞭1 200 bp的目的 嵌閤體RNA.N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P和pET-ms2-3V的錶達效率分彆為0.23、0.35、0.35和0.51 mg/ml.N-P3V-pET-C比N-P3V-pET-P錶達效率高52%,而pET-ms2-3V比P-3V-pET-P錶達效率高38%.所包裝的RNA具有耐RNase和DNase消化的特性以及良好的不同溫度條件下的穩定性.結論 通過改變噬菌體ms2 RNA包裝位點(19堿基的莖環結構)的數量,可構建能錶達內含達到理論上的約1 900 bp外源RNA的耐RNase病毒樣顆粒的原覈錶達載體平檯.通過應用包裝位點的變異體C-變異體,可以增加病毒樣顆粒的錶達效率.
목적 통과개변원서균체ms2포막단백RNA포장위점(19감기적경배결구)적수량급친화력,구건신적원핵표체계통,탐토표체내함장편단(체도이론상적1 900 bp)RNA적내RNase병독양과립적가능성.방법 수선설계함Hind Ⅲ화Not Ⅰ매절위점적인물,확증ms2포막단백적편마성숙매단백화의각단백적1 700 bp서렬,병장원래적19mer적포장위점서렬개변위C-5변이체(19 bp stem-loop결구중-5위적뇨밀정개변위포밀정),Hind Ⅲ화Not Ⅰ매절후,여용동양매절적표체재체pET-28(b)상련접,득도중조재체pET-ms2-pac.응용중첩PCR확증3충병독적5단감합체서렬(포괄3단SARS-CoV기인、일단HCV기인화일단HSN1기인),병재SARS-CoV3화HCV서렬지간삽입일개19mer적변이체포장위점서렬,재설계인물시,사감합체량단함유Not Ⅰ매절위점,여Not Ⅰ매절적중조재체pET-ms2-pac상련접,구건득도구유2개변이포장위점적표체재체pET-ms2-3V.동시구건3충대조중조표체재체,분별측정N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P화pET-ms2-3V 4충중조표체질립표체산물적260 nm흡광도(A260)치,근거공식A160=0.125 mg/ml계산4충표체산물적표체효솔.결과 성공구건료4충원핵표체재체:pET-ms2-3V、P-3V-pET-P、N-P3V-pET-P화N-P3V-pET-C.pET-ms2-3V화P-3V-pET-P경원핵표체후득도함전장위1 891적5단감합체RNA적병독양과립;N-P3V-pET-P、N-P3V-pET-C기원핵표체산물병독양과립중부포장료1 200 bp적목적 감합체RNA.N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P화pET-ms2-3V적표체효솔분별위0.23、0.35、0.35화0.51 mg/ml.N-P3V-pET-C비N-P3V-pET-P표체효솔고52%,이pET-ms2-3V비P-3V-pET-P표체효솔고38%.소포장적RNA구유내RNase화DNase소화적특성이급량호적불동온도조건하적은정성.결론 통과개변서균체ms2 RNA포장위점(19감기적경배결구)적수량,가구건능표체내함체도이론상적약1 900 bp외원RNA적내RNase병독양과립적원핵표체재체평태.통과응용포장위점적변이체C-변이체,가이증가병독양과립적표체효솔.
Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop.Methods 1.7 kb maturase and coat protein gene of ms2 were amplified.We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine.The 1700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac.The five target DNA sequences were spliced by overlapping extension(including 3 fragment of SARS-CoV,one fragment of HCV and one fragment of HSN1).PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM(R) T-easy vector and then excised with the Not Ⅰ restriction enzymes from the resulting recombinant plasmid.simoutaniously the pET-MC-pac plasmid was digested with Not Ⅰ restriction enzymes,fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V.Simoutaniously,we constructed three recombinant expression vector for control,including N-P3V-pET-P、N-P3V-pET-Cand P-3V-pET-P.N-P3V-pET-P contained one wild type pacsite located behind ms2 sequence:N-P3V-pET-C contained one C-variant pacsite located behind ms2 sequence:P-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence,another is located between SARS-CoV 3 and HCV sequence.Then these four expression vector were transformed into E.coli strain BL21(DE3),respectively.The 3V Armored RNA was expressed and purified.A260 absorbance value of expression product was determined.Results Four expression plasmids were constructed successfully.The Armored RNA with 1891 bases was snccessfully expressed by the two-paesite expression system of pET-ms2-3V and P-3V-pET-P;for N-P3V-pET-P、N-P3V-pET-C,only 1 200 bp target sequence was packaged into virus-like particles.Expression efficiency of N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P and N-P3V-pET-P was 0.23,0.35,0.35,0.51 mg/ml,respectively.Armored RNA was shown to have the charaeterization of Ribonuclease-resistant and stable at 4℃,37℃ and 25℃ respectively.Conclusion The expression plasmids containing two pacsites were constructed successfully and prokaryotic expression system can be used as an expressing plasmid platform for preparation of Ribonuclease-resistant virus-like particles containing long chimeric RNA sequence.
