重组家蚕病毒表达传染性法氏囊病病毒VP2蛋白

将传染性囊病病毒ＨＺ９６株主要宿主保护性抗原ＶＰ２的ｃＤＮＡ基因克隆到杆状病毒转移载体ｐＢａｃ－ＰＡＫ８中,获得重组转移载体ｐＢａｃＰＡＫ－ＶＰ２,载体ｐＢａｃＰＡＫ－ＶＰ２与修饰病毒Ｂｍ－ＢａｃＰＡＫ６线性化基因组ＤＮＡ共转染单层家蚕Ｂｏｍｂｙｘ ｍｏｒｉ（Ｂｍ）Ｎ细胞,经细胞内同源重组,筛选到重组病毒。ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ免疫鲩迹结果表明,ＶＰ２在家蚕培养细胞和家蚕幼虫中均得到了表达。     

鼠Ⅰ型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达

白细胞介素１（ＩＬ－１）是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素 １受体（ＩＬ－１Ｒ）结合而起作用。以杆状病毒为载体在昆虫细胞中克隆表达了小鼠Ｉ型可溶性白细胞介素１受体（ｓＩＬ－１　ＲＩ）基因。以ＮＩＨ／３Ｔ３细胞ＲＮＡ为模板,采用ＲＴ－ＰＣＲ方法扩增得到小鼠ｓＩＬ－ＩＲＩ的ｃＤＮＡ,克隆至杆状病毒转移载体ｐＡｃＧＰ６７Ｂ,将转移重组质粒与野生病毒ＡＣＮＰＶ ＤＮＡ共转染昆虫细胞Ｓｆ９,经同源重组得到重组杆状病毒ｒＡＣＮＰＶ。应用经纯化的ｒＡｃＮＰＶ感染昆虫细胞Ｓｆ９,表达获得重组的ｓＩＬ－１ＲＩ。经对亲和层析样品的ＳＤＳ－ＰＡＧＥ分析和对ＩＬ－１β生物活性阻断作用实验证实,表达产物能够与其配基结合,并且能够分泌至细胞培养上清中。     

鼠I型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达

白细胞介素１（ＩＬ－１）是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素１受体（ＩＬ－１Ｒ）结合而起作用,以杆状病毒为载体在昆虫细胞中克隆表达了小鼠Ｉ型可溶性白细胞介素１受体基因,以ＮＩＨ／３Ｔ３细胞ＲＮＡ为模板,采用ＲＴ－ＰＣＲ方法扩增得到小鼠ｓＩＬ－１ＲＩ的ｃＤＮＡ,克隆至杆状病毒转移载体ｐＡｃＧＰ６７Ｂ,将转移重组质粒与野生病毒ＡｃＮＰＶ ＤＮＡ共转染昆虫细胞Ｓｆ９,     

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）。     

重组腺病毒介导的白细胞介素—12（IL—12）在肝癌细胞中的表达

利用腺病毒表达系统在肝癌细胞中成功地表达了有生物活性的白细胞介素－１２（ＩＬ－１２）。ＩＬ－１２的Ｐ３５和Ｐ４０ ｃＤＮＡ分别克隆到腺病毒载体ｐＡＣＣＭＶ．ｐＬｐＡ,构建ｐＡＣ／Ｐ３５和ｐＡＣ／Ｐ４０表面质粒。与腺病毒重组质粒ｐＪＭ１７共转染２９３细胞,通过基因重组产生ＩＬ－１２Ｐ３５和Ｐ４０重组腺病毒。用重组腺病毒感染肝癌细胞株ＨｅｐＧ２和ＳＭＭＣ７７２１,经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ检测证明,     

hBMP—2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高,细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃ     

大鼠酰胺化酶的信号肽引导的昆虫细胞表达外泌系统

将大鼠酰胺化酶的信号肽及前导肽编码序列引入昆虫核多角体病毒转移表达载体,构建ＰＡＢＣｈＧＲＦ（Ｇｌｙ）、ＰＡＢＣＩＧＦＩ融合基因的昆虫细胞分泌表达质粒ｐＢａｃＰＡＧ２、ｐＢａｃＰＡＩ,并与经修饰的银纹夜蛾核多角体病毒ＢａｃＰＡＫ６线性化ＤＮＡ共转染秋粘虫细胞Ｓｆ２１,通过同源重组、筛选和鉴定,得到它们的重组病毒ＢａｃＰＡＧ、ＢａｃＰＡＩ。将重组病毒感染Ｓｆ２１细胞,ＰＡＢＣｈＧＲＦ（Ｇｌｙ）和ＰＡＢＣＩＧＦＩ均得到有效外泌表达,表达产物通过ＩｇＧＳｅｐｈａｒｏｓｅ柱可获得快速纯化。     

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。     

hBMP－2cDNA在COS细胞和小鼠肌肉中的表达

研究了骨形态发生蛋白ＢＭＰ－２ｃＤＮＡ在ＣＯＳ细胞和小鼠肌肉中的表达的情况,从ｐＳＰＳ６５ＢＭＰ－２质粒中回收ＢＭＰ－２ｃＤＮＡ,删除５＇端的非翻译序列,插入ｐＳＶＬ载体中,构建了含有ＢＭＰ－２全长编码序列的重组表达质粒ｐＳＶＬＢＭＰ－２。将表达质粒导入ＣＯＳ－７细胞中,细胞ＲＮＡ点杂交结果表明,转染ＢＭＰ－２基因的细胞内ＢＭＰ－２的ｍＲＮＡ水平明显升高;细胞培养上清的ＥＬＩＳＡ显示,转染ＢＭＰ－２ｃＤＮＡ后,细胞分泌产生的ＢＭＰ－２显著增加。小鼠实验发现,在肌肉内用注射法导入ＢＭＰ－２重组质粒后,局部组织内ＢＭＰ－２的ｍＲＮＡ转录水平也明显提高。     

PAI—2抑制细胞凋亡依赖其蛋白质结合功能域

将纤深酶原激活剂抑制物２型（ＰＡＩ－２）突变体ＰＡＩ－２ＣＤ和ＰＡＩ－２ＱｃＤＮＡ与真核表达载体ｐｃＤＮＡ３重组,构建真核表达质粒并转染ＨｅＬａ细胞,经Ｇ４１８筛选,Ｎｏｒｔｈｅｒｎ迷鉴定和ＥＬＩＳＡ检测,筛选出稳定表达ＰＡＩ－２ＣＤ和ＰＡＩ－２Ｑ的阳性细胞株。ＥＬＩＳＡ结果表明ＰＡＩ－２突变蛋白质在转染细胞中的表达量与相应野生型ＰＡＩ－２在阳性细胞克隆中的表达量基本相近,ＰＡＩ－２及其突变体在理     

用杆状病毒为载体在昆虫细胞中表达马立克病病毒pp38基因

鸡马立克病病毒(MDV)38kd磷蛋白(pp38)基因中包括起始密码子和终止密码子的完整编码序列被整合进杆状病毒AcNPV的转移载体质粒pVL1392,用所得的含pp38基因的重组转移载体质粒pVLpp38I与野生型杆状病毒AcNPV的DNA共转染昆虫传代细胞系Sf9细胞后,用荧光抗体法以抗MDV单克隆抗体H_(19)筛选到能表达MDVpp38的重组杆状病毒克隆BP38 I。免疫印迹试验表明,在重组病毒BP38 I感染的Sf9细胞溶解物中,可表现一条分子量约为35—36kd的为单克隆抗体H_(19)识别的MDV特异性蛋白带。     

High level expression of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator in silkworm

A cDNA of a mutant (K151E, R154G) of single chain urokinase-type plasminogen activator (mscu-PA) was constructed to include the natural scu-PA signal peptide sequences and transferred into the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) by transfer vectors pBE284 (derived from BmNPV) and pVL1392 (from AcNPV), respectively. Both Bombyx mori (BmN) cells and silkworm larvae were infected with the two recombinant viruses. Fibrin-plate assay showed that the re-virus from pVL1392 increased the yield of mscu-PA three times compared with the re-virus from pBE284.     

重组人卵泡刺激素在昆虫细胞中的表达

为了研究在昆虫细胞中表达重组人卵泡刺激素,我们以人胎盘组织提取的染色体DNA为模板,利用重叠PCR方法扩增出hFSHβ亚基的cDNA的编码区。将此cDNA克隆入核型多角体病毒(AcNPV)非融合蛋白基因表达载体pVLl393,我们得到了表达载体pVLl393-hFSHβ,然后与BaculoGold^TM线性杆状病毒DNA共转染昆虫细胞SF9,经多次扩增后获得高滴度的重组病毒AcNPV-hFSHβ。将此重组病毒感染昆虫细胞,我们得到了在胞浆中表达的hFSHβ亚基,Western blot显示分子量大约为21kDa。以重组病毒AcNPV-hFSHβ与AcNPV-hCGoL一同感染昆虫细胞得到了具有分泌性的重组hFSH异二聚体,在非还原的条件下Western blot显示分子量大约为33kDa。     

Purification of pancreatic cholesterol esterase expressed in recombinant baculovirus-infected Sf9 cells.

A cDNA clone encoding the entire coding sequence of rat pancreatic cholesterol esterase (bile salt-stimulated lipase) was subcloned into the Baculovirus transfer vector pVL1392 and used to co-transfect Spodoptera frugiperda (Sf9) insect cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. Two recombinant proteins (M(r) 74 kDa and 64 kDa) reactive with anti-cholesterol esterase IgG were produced and secreted by the infected Sf9 cells in large quantities in a time-dependent manner. The 74-kDa protein was detectable in the cultured medium at the second day post-infection and increased progressively, reaching a level of 50 micrograms/ml of culture medium after 8 days. Amino-terminal sequencing of this recombinant protein showed that the signal peptide of cholesterol esterase was correctly cleaved, resulting in the production of mature protein. The 64-kDa recombinant protein was not detected in the medium until Day 5 post-infection and accumulated to a level of 25 micrograms/ml at Day 8. Both the 74- and the 64-kDa cholesterol esterases were biologically active and hydrolyzed the artificial substrate p-nitrophenyl butyrate. Results of this study demonstrated that Baculovirus-infected Sf9 cells can be used for high-level expression of pancreatic cholesterol esterase. The recombinant enzyme will be useful for further characterization of this protein.     

人绒毛膜促性腺激素β亚基和绵羊垂体促性腺激素共有α亚基组成的单链多...

hCG beta-oLH alpha chimeric cDNA was constructed by using overlapping PCR to contact the codons of C-terminal end of hCG beta with the codons of N-terminal end of oLH alpha, then it was subcloned into nuclear polyhedrosis virus (AcNPV) expression vector pVL1393 to construct expression vector pVL1393-hCG beta-oLH alpha. The insect cells (Sf9) were cotransfected by the expression vector pVL1393-hCG beta-oLH alpha and BaculoGold AcNPV linearized genomic DNA, and recombinant viruses AcNPV-hCG beta-oLH alpha were screened out by plaque assay. Further the insect cells were infected by the recombinant viruses, the recombinant hCG beta-oLH alpha was purified by immunoaffinity chromatography column coupling anti-hCG beta monoclonal antibody from the conditioned media of infected cells. The results of SDS-PAGE silver staining and western blotting showed that hCG beta-oLH alpha single peptide chain had apparent molecular weights of 40.5 kD and 38.0 kD under non-reducing and reducing conditions respectively, indicating the occurrence of disulfide bonds and significant tertiary structure in the single peptide chain. From the results of competitive inhibition of 125I-hCG beta binding we can conclude that the anti-hCG beta antibody-binding activity of hCG beta-oLH alpha chimera is lower than that of native hCG, but higher than that of native hCG beta. Therefore, we assume that the hCG beta-oLH alpha chimera should have potential application as a target antigen of anti-hCG fertility regulatory vaccine.     

人绒毛膜促性腺激素β亚基和补体C3d片段组成的单链多肽的生物合成

In view of the strong immunity-enhancing function of HEL-C3d3 designed by Dr. Paul W. Dempsey, we made our efforts to produce a similar recombinant protein of hCG beta. With polymerase chain reaction, we introduced a Bam HI restriction site into the 3' terminal of hCG beta cDNA. The new cDNA and its terminal's correctness has been confirmed by sequencing. Then we have it covalently attached to the C3d3 cDNA at the pre-designed Bam HI/Bgl II site. Having the chimeric DNA correctly cloned into the protein nuclear polyhedrosis virus (AcNPV) expression vector pVL1393, we constructed the expression vector pVL1393-(hCG beta-C3d3). The insect cells were co-transfected with the expression vector and linearized nuclear polyhedrosis virus DNA, and recombinant viruses AcNPV-(hCG beta-C3d3) were screened out. Through anti-hCG beta immunoaffnity chromatography, the recombinant hCG beta-C3d3 chimera polypeptide was purified from culture supernatant of insect cells infected by the recombinant viruses. In RIA test, the expressed product competitively inhibits the binding of 125I-hCG beta to hCG beta-antibody. On SDS-PAGE and Western blot, the recombinant peptide hCG beta-C3d3 obviously appears to be with a molecular weight of 116KD. Therefore, we arrive at a conclusion that it has a normal immunogenic ability.     

Bruton酪氨酸激酶在昆虫细胞中表达的初步研究

用杆状病毒表达载体系统表达小鼠Ｂｒｕｔｏｎ酪氨酸激酶（Ｂｒｕｔｏｎｔｙｒｏｓｉｎｅｋｉｎａｓｅ,Ｂｔｋ）．构建重组转染载体时,于Ｂｔｋ起始码的上游插入了一段Ｈ９０２序列．用重组转染载体、苜蓿夜蛾核型多角体病毒线性ＤＮＡ和质脂体共转染Ｓｆ９昆虫细胞,经过对重组杆状病毒三轮扩增后,用Ｈ９０２抗体检测表明,昆虫细胞中Ｂｔｋ的表达已达最高水平．对表达后Ｂｔｋ自身磷酸化检测表明,该激酶具有自身磷酸化活性．从而证实Ｂｔｋ在昆虫细胞中的表达获得了成功     

Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus

Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393. Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of SINPV. The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs. Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of β-galactosidase (βGal). The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable. The highest βGal activity was obtained with S1AcNPV4.lacZ. Production of βGal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ. These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses.     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。