苜蓿丫纹夜蛾核型多角体病毒(AcMNPV)大立方形多角体突变株的分子生物学研究

利用空斑技术,从既能形成多角体又含ＴＫ酶基因的苜蓿丫纹夜蛾重组核型多角体病毒ＡｃＭＮＰＶ－ＴＫ中,纯化了一株形成大立方形多角体的病毒突变株ＡｃＭＮＰＶ－ＴＫｍｔ５１３。用ＥｃｏＲＩ、ＰｓｔⅠ、ＢｇｌⅡ及ＫｐｎⅠ等限制性内切酶对它做了酶切分析,并克隆了多角体蛋白全基因及其侧翼部分的片段。对所克隆的部分全序列测定结果,发现在多角体蛋白基因读码框内只出现了一个碱基的突变,导致了第２５位的氨基酸密码子由ＧＧＴ变为ＧＡＴ,该位氨基酸由甘氨酸（Ｇｌｙ）变为天冬氨酸（Ａｓｐｑ）还利用ＰＣＲ技术扩增出突变了的多角体蛋白基因部分片段,并将它克隆入转移载体质粒ｐＥＶ５５,与不形成多角体的重组病毒ＴｎＮＰＶ－ＨＢｓＤ４ＤＮＡ共转染Ｓｆ９细胞,结果产生同样的大立方形多角体病毒。     

重组人干细胞因子在昆虫细胞中的高效表达

含信号肽的可溶性人干细胞因子（ｈＳＣＦ）ｃＤＮＡ 基因重组于杆状病毒转移载体ｐＶＬ９４１ 中,重组转移载体ｐＶＬ９４１ＳＣＦ与野生型苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）ＤＮＡ 共转染草地夜蛾细胞Ｓｆ９ 后,通过体内同源重组构建了重组病毒ＡｃＮＰＶＳＣＦ。Ｓｏｕｔｈｅｒｎ 杂交表明重组病毒基因组中含有ｈＳＣＦ基因片段。重组病毒感染单层Ｓｆ９ 细胞后,表达产物分泌到胞外培养液中。用ＭＴＴ 比色法和ＴＦ１ 细胞株测定表达产物与ＩＬ３ 的协同效应,测得感染重组病毒的培养细胞第三天表达量为１９７０ ｕｎｉｔｓ／ｍ Ｌ培养液。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ 分析可见分子量为１８ ×１０３ 、２０ ×１０３ 和２２ ×１０３ 三条带。     

黄地老虎颗粒体病毒DNA片段的克隆与颗粒体蛋白基因的定位

用ｐＵＣ１９质粒作载体,克隆了黄地老虎颗粒体病毒（Ａｇｒｏｌｉｓｓｅｇｅｔｕｍｇｒａｎｕｌｏｓｉｓｖｉｒｕｓ,简称ＡｓＧＶ）ＤＮＡＰｓｔＩ－Ｄ．Ｅ．Ｆ．Ｇ．Ｈ．Ｊ．Ｋ．等７个片段。以［￣（３２）Ｐ］－ｄＣＴＰ标记的油桐尺蠖核型多角体病毒（Ｂｕｚｕｒａｓｕｐｐｒｅｓｓａｒｉａｎｕｃｌｃａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ简称ＢｓＮＰＶ）多角体蛋白基因为探针,在３７℃条件下对ＡｓＧＶ）颗粒体蛋白基因进行了定位,将其分别定位于ＢｓｌⅡ－Ｓ或ＴＰｓＴＩ－Ａ或Ｂ和ＥｃｉＲＩ－Ａ片段上。     

猪生长激素基因在杆状病毒载体系统中的表达

通过对猪生长激素（ｐＧＨ）基因的ｃＤＮＡ进行测序,得到ｐＧＨｃＤＮＡ的全序列,并与Ｓｅｅｂｕｒｇ等报道的序列进行了比较和讨论。然后利用具人工合成启动子和多角体蛋白ＸＩＶ启动子的转移载体质粒ｐＳＸＩＶＶＩ＾＋Ｘ３／４构建出含ｐＧＨ基因的重组质粒ｐＸ３／４－ｐＧＨ。将ｐＸ３／４－ｐＧＨ与致死缺失型线性化ＡｃＭＮＰＶ－ＯＣＣ＾－ＤＮＡ共转染Ｓｆ９细胞,构建出既能形成多角体又能表达ｐＧＨ基因的苜蓿丫纹夜蛾     

昆虫杆状病毒衣壳主蛋白基因的PCR扩增,克隆和定位

用ＰＣＲ技术成功地扩增了苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的衣壳主蛋白基因（ｖｐ３９基因）,并克隆了该基因,利用纯的ｖｐ３９基因探针,在低严谨杂交条件下,已将粘虫核型多角体病毒（ＬｓＮＰＶ）的ｖｐ３９基固定位在ＰｓｔＩ－Ｆ,ＢａｍＨＩ－Ｃ,ＥｃｏＲＩ－Ｃ,ＸｈｏＩ－Ｄ,Ｉ,ＥｃｏＲＶ－Ｈ,Ｘ等片段上。ＰＣＲ反应时,在扩增出预期的包括完整ｖｐ３９基因的１４０６ｂｐ片段的同时也扩增出一条Ｃａ．４００ｂｐ的片段,本文讨论了ＰＣＲ的特异性扩增和非特异性扩增。     

中国棉铃虫核型多角体病毒几丁质酶基因的定位与克隆

以α３２ＰｄＡＴＰ标记含ＣｆＭＮＰＶ几丁质酶基因的重组质粒为探针,在６８℃条件下对棉铃虫单粒包埋核型多角体病毒（ＨａＳＮＰＶ）进行Ｓｏｕｔｈｅｒｎ杂交,将ＨａＳＮＰＶ的几丁质酶基因分别定位在ＢａｍＨＩＥ、ＢｇｌⅡＥ、ＥｃｏＲＩＧ、ＨｉｎｄⅢＦ、ＸｂａＩＨ、ＢａｍＨＩ＋ＨｉｎｄＩＩＭ和ＢａｍＨＩ＋ＸｂａＩＨ,并以ｐＴＺ１９Ｒ为载体获得了ＸｂａＩＨ片段克隆。     

茶尺蠖核型多角体病毒多角体蛋白基因核苷酸序列及其在Baculoviridae中的地位(英文)

茶尺蠖核型多角体病毒（ＥｏＳＮＰＶ）基因组的ｐｏｌｈ和ｅｇｔ基因区约１４．２ｋｂ的酶切图谱被构建．ｅｇｔ基因位于ｐｏｌｈ基因上游约４．８ｋｂ处,但转录方向与ｐｏｌｈ基因相反．ＥｃｏＲⅤ－Ｌ片段ｐｏｌｈ基因及其旁侧的１１２５核苷酸序列被测定．ｐｏｌｈ基因编码区长７３８核苷酸,可编码２４６氨基酸的多肽．起始密码子ＡＴＧ上游是一个富含ＡＴ（ＡＴ占７１．２％）的启动子区,在－５２核苷酸处有杆状病毒晚期基因启动子转录起始基序ＡＴＡＡＧ．在终止密码子下游２０８核苷酸有一个ｐｏｌｙ（Ａ）信号,ＡＡＴＡＡＡ．但ＥｏＳＮＰＶｐｏｌｈ基因起始密码子ＡＴＧ相邻核苷酸序列为ＧＴＡＡＴＧＴ,其－３是个Ｇ,这与已知的１６种其它杆状病毒ｐｏｌｈ基因－３位置均是Ａ不相同．在分析了ＥｏＳＮＰＶ和ＨａＳＮＰＶ多角体蛋白基因核苷酸序列的基础上,通过ＭＡＬＩＧＮ程序,比较了目前已发表的２６种杆状病毒包涵体蛋白的序列,ＥｏＳＮＰＶ与黄杉毒蛾核型多角体病毒（ＯｐＳＮＰＶ）的同源性为最高,核苷酸序列的同源性为８３．０％,氨基酸序列达９４．７％;与其它２０种鳞翅目ＮＰＶ的同源性也很高,核苷酸序列同源性为７２．６％～８１．９％,氨基酸序列为８３．７％～９３     

粘虫核型多角体病毒多角体蛋白基因结构和序列的研究

测定了粘虫核型多角体病毒（Ｌｅｕｃａｎｉａｓｅｐａｒａｔａｎｕｃｌｅａｒｐｏｌｙｈｅｄｒｏｓｉｓｖｉｒｕｓ,ＬｓＮＰＶ）两个ＥｃｏＲＶ片段的共１２０１ｂｐ序列,发现了一个７１４ｂｐ的开放阅读框（ＯＲＦ）,根据其与多种ＮＰＶ多角体蛋白基因（ｏｃｕ）同源性的比较和５＇端典型的１４ｂｐ保守序列,说明此ＯＲＦ即为ＬｓＮＰＶ的ｏｃｕ基因。根据核苷酸序列预测的多角体蛋白有２４６个氨基酸,其中酸性和碱性氨基酸数相等,疏水氨基酸含量很高,氨基酸组成中以谷氨酸（Ｇｌｕ）含量最高,谷氨酰胺和半胱氨酸含量最低。编码区碱基同源性与ＭｂＮＰＶ最高,为９７．０％;氨基酸同源性与ＭｂＮＰＶ最高,为９７．５％。密码子偏爱选用以第三个碱基为嘧啶的密码子。二级结构分析表明,α－螺旋（Ｈ）和β－折叠（Ｓ）相等,β－转角含量是Ｈ和Ｓ含量之和。     

重组人GM—CSF基因在昆虫细胞中的表达

利用苜蓿夜蛾核型多角体病毒（ＡｃＮＰＶ）带β－Ｇａｌａｃｔｏｓｉｄａｓｅ基因标记的非融合蛋白基因转移载体ｐＢｌｕｅＢａｃ将人粒细胞巨噬细胞集落刺激因子（ｈＧＭ－ＣＳＦ）基因成功地插入病毒ＡｃＮＰＶ的基因组中．ｈＧＭ－ＣＳＦ基因在感染重组病毒的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ）培养细胞Ｓｆ９中得到表达,感染后的Ｓｆ９细胞培养液能刺激人骨髓细胞在体外形成典型的集落,表达水平可达２．７×１０５５ＣＦＵ／ｍｌ。以ｈＧＭ－ＣＳＦ单抗所作的ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ表明,表达的ｈＧＭ－ＣＳＦ对是３种糖基化程度不同的产物,分子量分别约为１５ｋｄ,１８ｋｄ和２０ｋｄ。     

人生长激素基因在昆虫细胞中的表达(英文)

近年发展起来的昆虫细胞－杆状病毒基因工程系统具有良好的优越性：（１）表达产量高,（２）产物的免疫原性、功能性类似于天然产物,（３）适用于悬浮及无血清培养。本实验中,将外源基因ｈＧＨ克隆至质粒ｐＶＬ１３９３,形成转移载体ｐＶＬ１３９３／ｈＧＨ（Ｆｉｇ．１＆２）。与昆虫核多角体病毒ＡｃＮＰＶ基因组ＤＮＡ共转染秋粘虫细胞Ｓｆ９（Ｆｉｇ．３）,ｐＶＬ１３９３／ｈＧＨ与ＡｃＮＰＶ基因组ＤＮＡ发生同源重组,取代多角体蛋白基因。经空斑分析纯化得到不含多角体,单一表达的重组病毒ｒＡｃＶｈＧＨ（Ｆｉｇ．４）,感染滴度达到２．０３ｘ１０８ＰＵＦ／ｍＬ。经反复实验,１０５细胞／ｍＬ感染６～７天后收获培养上清液,可得到表达量为４０μｇ／ｍＬ的ｈＧＨ蛋白（Ｆｉｇ．５,６＆７）。     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.     

汉坦病毒囊膜糖蛋白G1基因重组腺病毒载体的构建及其在Vero E6细胞中的表达

拟构建汉坦病毒Gl基因重组腺病毒载体并在VeroE6细胞中表达,为汉坦病毒基因疫苗的研究提供实验基础。PCR法从含汉坦病毒-76118株M基因的M56质粒扩增糖蛋白G1基因片段,利用穿梭质粒pShuttle,将其克隆入Adeno—X病毒DNA,获得重组腺病毒DNA,转染HEK293细胞,包装、扩增后得到汉坦病毒Gl基因重组腺病毒原种,感染VetoE6细胞,用IFA法和ELISA法检测表达产物。得到了含汉坦病毒G1基因的重组腺病毒,其滴度约为10^11pfu／ml,感染VeroE6细胞后检测到汉坦病毒糖蛋白G1的表达。     

密码子偏性对痘苗病毒载体表达效率影响的研究

为了研究密码子偏性对痘苗病毒载体表达效率的影响,分别采用痘苗病毒及其宿主细胞的优势密码子对绿色荧光蛋白基因进行改造,利用荧光、Western blot和FCM等方法分析其在痘苗病毒载体系统的表达水平。结果显示,全部采用痘苗病毒优势密码子(富含A T)和全部采用宿主细胞优势密码子(富含G C),以及部分使用宿主细胞优势密码子的三种绿色荧光蛋白基因都能够有效表达,表达水平相近,表明痘苗病毒载体对目的基因密码子的使用具有很好宽容性。为了探讨这种宽容性的机理,分别利用在胞核内和在胞浆内转录的质粒载体对不同密码子偏性的绿色荧光蛋白基因进行表达分析。结果显示,胞核内转录目的基因的pcDNA3质粒载体能有效表达富含G C的绿色荧光蛋白基因,不能有效表达富含A T的绿色荧光蛋白基因,而胞浆内转录目的基因的pSCA质粒载体能同样有效表达上述不同密码子偏性的目的基因。这些结果表明,位于胞浆内的富含A U的转录产物能够有效表达,细胞核内生成的富含A U的转录产物可能受核膜屏障或其它核内因素影响而不能有效表达。因此,胞浆内繁殖的特性是痘苗病毒载体具有密码子宽容性的主要原因。此研究为痘苗病毒载体和常用真核表达载体的选择使用提供了重要实验依据。     

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3'' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.     

病毒诱导番茄的基因沉默

该实验通过RT-PCR获得了番茄八氢番茄红素脱氢酶(Phytoene desaturase,PDS)基因的部分序列,双酶切PDS片段和烟草脆裂病毒载体(pTV00),构建重组载体pTV00-PDS,经农杆菌GV3101介导侵染番茄叶片并观察植株表型变化.结果显示,被侵染的番茄表现出明显的光漂白现象.半定量RT-PCR检测表明,PDS的mRNA被显著降解.该沉默体系的建立为下一步大规模验证番茄基因功能奠定了基础.     

昆虫杆状病毒系统表达口蹄疫病毒3ABC基因

以O型口蹄疫病毒为研究对象,经过RTPCP扩增得到非结构蛋白3ABC基因,克隆到转移载体pFastbacHT,将其转入含穿梭载体Bacmid的DH10Bac,与Bacmid发生位点特异性转座作用,得到3ABC的重组穿梭载体Bacmid3ABC,再将其转染昆虫细胞HiFive。PCR鉴定证实3ABC基因正确地插入到病毒基因组的多角体蛋白基因启动子下游,经过SDSPAGE和Westernblot检测,3ABC基因在昆虫细胞中表达了大小约为50kDa的蛋白条带,3ABC基因在BactoBac系统中的成功表达为建立以基因工程产品为抗原、鉴别诊断自然感染和免疫动物的方法提供了技术条件。     

Expression of foreign gene by cysteine proteinase null recombinant baculovirus

The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.     

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.     

甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

利用DNA重组技术,将甜菜坏死黄脉病毒(BNYVV)内蒙分离物的CP基因和54kDa通读区片段拼接。构建了BNYVV 75kDa通读蛋白基因。序列分析表明,构建的75kDa通读蛋白基因与野生型相比．只有4个核苷酸发生了改变(包括将CP基因的终止密码子TAG改造为ATG),相应地2个氨基酸也发生了改变。将75kDa通读蛋白基因及其54kDa片段分别克隆到pJw2上,构建了这两十基因的原核表达载体。SDS—PAGE和western blotting检测结果表明,75kDa通读蛋白基因在E coli BL21(DE3)中经温度(42℃)诱导后除可特异地表达75kDa蛋白外。还产生两种小蛋白。75kDa通读蛋白基因的54kDa片段只表达出37kDa的蛋白。