伪狂犬病病毒闽A株gE基因去信号肽片段在Pichia pastoris中的表达

伪狂犬病病毒囊膜糖蛋白E是一种在伪狂犬病根除计划中具有重要作用的糖蛋白.将伪狂犬病病毒闽A株gE基因去信号肽片段克隆到巴斯德毕赤酵母（Pichia pastoris）表达载体pPICZαA中,获得的重组表达载体pPICZαA-FL电击转化野生型酵母菌SMD1168后,得到多株酵母工程菌SMD1168/pPICZαA-FL.经高浓度Zeocin^TM筛选、表型鉴定、工程菌的诱导表达及表达产物的鉴定,最后得到高效表达gE基因去信号肽片段的酵母工程菌SMD1168/pPICZαA-FL-7.工程菌72 h培养上清的SDS-聚丙烯酰胺凝胶电泳与蛋白质印迹结果显示,gE基因去信号肽片段表达产物大小约为80 ku,比预期的63.8 ku大.凝胶薄层扫描结合Bradford蛋白质总含量测定结果表明,表达产物占工程菌培养上清总蛋白的13.49%,表达量可达11.7 mg/L.间接ELISA结果表明重组表达产物具有良好的抗原性,能够有效地区分伪狂犬病病毒gE标准阳性与阴性血清.     

鹅源新城疫病毒NP、P和L基因的克隆与P基因的表达鉴定

将鹅源新城疫病毒的NP、P和L基因通过RT PCR方法从尿囊液中扩增后分别克隆进pGEM-Teasy载体 ,再分别亚克隆到真核表达载体pCI neo上 ,通过酶切、PCR和测序验证克隆正确。利用P基因开放性阅读框 (ORF)上靠近终止密码上游的AgeI位点 ,将报告基因绿色荧光蛋白 (GFP)基因克隆进P基因真核表达重组质粒 ,分别转染COS-1细胞和CEF细胞 ,在倒置荧光显微镜下可见到绿色荧光 ,表明GFP基因已得到表达 ,由此证明P相似文献   

一种高R-选择性水解R,S-甲霜灵的酯酶基因的克隆表达及表征

[目的] 将一种可以高选择性水解R-甲霜灵的新脂酶基因,在大肠杆菌中进行克隆和表达,并研究重组脂酶的性质。[方法] 根据已知的目标酯酶N端10个氨基酸序列,在已测序的Albibacters sp. zjut528基因组中找到相匹配的一个酯酶基因,它全长969 bp,编码322个氨基酸,将该基因命名为RMest。通过引物扩增得到该基因的DNA片段,将它与表达载体pET-28a（+）连接后,转化大肠杆菌BL21Gold（DE3）,构建重组菌,IPTG诱导表达该酯酶,并用Ni²⁺亲和层析介质进行纯化。[结果] 在重组菌RMest-pET-28a（+）-E.coli BL21 Gold（DE3）中成功表达了重组酯酶RMesterase,大小约为46 kDa。用胞内重组酶液催化水解R,S-甲霜灵,底物浓度10 g/L,反应6 h,底物转化率为49.8%,产物（甲霜灵酸）的ee_p为99.3%,对底物的对映体R-构型具有专一（选择）性。该酶最适温度和pH分别为40℃和pH 9.0。该酶的活性受到产物甲醇的抑制。通过Blast+在Uniprot KB数据库中搜寻与酯酶RMest同源的蛋白,采用邻近法构建该酶的蛋白系统发育树,结果显示它与某些Lysophospholipase、AB hydrolase-1 domain-containing protein和Esterase的同源性最高,但是与它们均存在较大的进化距离,表明该酶是一种相对独立进化的新酯酶。[结论] 在大肠杆菌中成功克隆和表达了一种新的脂酶基因RMest,重组酯酶RMesterase可以高手性选择性水解R,S-甲霜灵生成R-甲霜灵酸。     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNA Bac-N-Blue^TM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。     

口蹄疫病毒NSP 3ABC基因在昆虫细胞中的分泌表达及其活性检测

用设计的特异引物,扩增得到了N-端带有6×His编码序列的口蹄疫病毒完整3ABC基因序列,并将其亚克隆入带有蜂毒溶血肽序列的穿梭质粒pMelBac-B中,构建了重组质粒pMel-3ABC。将该重组质粒与杆状病毒骨架DNA Bac-N-Blue^TM共转染Sf9昆虫细胞,通过噬斑筛选和PCR鉴定,获得了含有目的基因的重组杆状病毒。重组病毒感染Sf9昆虫细胞,采用通过SDS-PAGE和Western blot检测,证明目的基因在昆虫细胞中得到了正确的表达,表达产物分泌至细胞培养上清中,并具有良好的生物活性。表达的目的蛋白经过镍柱亲和层析法纯化后,用间接ELISA方法检测与口蹄疫病毒感染动物血清的反应性,证明表达目的蛋白与感染动物血清有很好的反应性而与正常动物以及免疫动物血清不发生反应。该研究为建立一种更加敏感和特异的口蹄疫病毒感染动物与疫苗免疫动物的鉴别诊断方法奠定了基础。     

伪狂犬病病毒Fa株胸苷激酶基因缺失株的构建

采用外切除缺失法对已克隆于ｐＢＲ３２２中包含伪狂犬病病毒（ＰＲＶ）胸苷激酶（ＴＫ）基因的ＢａｍＨＩ－１１片段（ｐＰＢ１１）进行改建,经筛选获得４株ＴＫ基因缺失重组质粒（ｐＤＴＫ３－１．ｐＤＴＫ３－６,ｐｄＴＫ３－８和ｐＤＴＫ５－６）对其进行酶切鉴定和Ｓｏｕｔｈｅｒｎ转印杂交,结果其１１片段迁移率均发生改变,缺失的碱基数分别约１２５０,１１００,１２５０和２００ｂｐ,用ＰＲＶＦａ－ＤＮＡ或ＰＲＶＦａ     

cry3A和vhb基因在转基因马铃薯中的表达

分别构建了含cry3A和cry3A+vhb基因的植物表达载体pBCry3A和pBC₃Vhb,并通过根癌农杆菌介导转化了马铃薯. 对转化再生植株进行PCR和DNA印迹分析表明,外源基因已整合到马铃薯基因组中, 且连续三代无性繁殖后转基因仍存在. ELISA分析表明cry3A基因在转基因植株中得到了高效表达, 在单转cry3A植株中最高表达量达0.1%, 转cry3A与vhb双基因株系中为0.065%. 水涝试验显示,转双基因且vhb mRNA的RT-PCR呈阳性的马铃薯植株,对低氧胁迫有较好的耐受性, 表明获得的上述转双基因马铃薯株系可能会具有很好的抗虫和耐涝性能.     

表达猪圆环病毒Ⅱ型ORF2基因的重组猪伪狂犬病病毒的免疫原性

【目的】为研制预防猪圆环病毒II型(PCV2)感染的重组伪狂犬病病毒(PRV)活载体疫苗。【方法】将PCV2 ORF2基因插入到PRV通用载体pG中,利用脂质体LipofectamineTM2000试剂盒将重组转移质粒pGO与猪PRV弱毒HB98株DNA共转染猪睾丸(ST)细胞,通过3轮蚀斑纯化重组病毒。将重组病毒、商品化PCV2灭活苗及DMEM培养液分别免疫6周龄雌性昆明小鼠,4周后加强免疫1次,首免后第8周用PCV2强毒NY株对小鼠进行攻毒。【结果】成功获得表达ORF2基因的重组病毒PGO,首免重组病毒后小鼠体内抗PCV2的ELISA抗体水平很低,二免后小鼠PCV2特异的ELISA抗体水平明显升高,并且重组病毒组能够激发PCV2特异的淋巴细胞增殖效应。攻毒试验表明重组病毒组和PCV2灭活疫苗组均能有效抵抗PCV2强毒攻击。【结论】表明表达ORF2基因的重组病毒PGO具有良好免疫原性。     

转新型抗虫基因Vip3A棉花新种质的创制

该研究构建植物表达载体pBin438 Vip3A,通过农杆菌介导法转化棉花品种‘冀合713’,将新型抗虫基因Vip3A导入到棉花植株,创制对棉铃虫抗性的转基因棉花新种质。结果表明：(1)PCR检测Vip3A基因已经导入到棉花基因组中且能够稳定遗传。(2)室内抗虫性鉴定表明,与对照相比转基因植株对棉铃虫的抗性显著提高,并获得2株高抗和3株抗棉铃虫的转Vip3A基因株系。(3)Southern blotting结果显示,转基因株系BV01为单拷贝。(4)Elisa检测表明,外源Vip3A基因在BV01的根、茎、叶、花、种子中都有表达,在叶片中的Vip3A蛋白表达为苗期>蕾期>花期>铃期>吐絮期。该研究创制了新型抗虫转基因棉花材料,为培育棉花新型抗虫品种提供了种质资源。     

Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

表达猪瘟病毒E2蛋白的BacMam病毒的构建及其免疫原性分析

含具有哺乳动物细胞活性的启动子的重组杆状病毒(BacMam病毒)可有效转导多种哺乳动物细胞,并被广泛用于开发新型非复制型载体疫苗．将水泡性口炎病毒G蛋白(VSV-G)基因插入多角体启动子下游,得到经修饰的杆状病毒转移载体,将对虾白斑综合症病毒(WSSV)ie1启动子控制下的猪瘟病毒E2基因表达盒插入此载体中,构建了BacMam病毒BacMam/G-ie1-E2,以其感染Sf9细胞和转导HeLa细胞,通过间接免疫荧光试验和Western blot分析检测E蛋白的表达,同时用BacMam病毒直接免疫小鼠,用检测猪瘟病毒抗体的间接ELISA方法检测免疫小鼠血清抗体,用基于CFSE和WST-8的淋巴细胞增殖试验评价其细胞免疫应答．结果显示,BacMam/G-ie1-E2能同时在昆虫细胞和哺乳动物细胞中高效表达E2蛋白,免疫小鼠能诱导产生针对猪瘟病毒的特异性抗体,免疫小鼠脾细胞经猪瘟病毒刺激后能诱导特异性的淋巴细胞增殖．这表明,由BacMam病毒介导的基因转移有望用于开发针对猪瘟病毒的非复制型载体疫苗．     

Sequence complexity of the S receptor kinase gene family in Brassica

A genomic library from an S ₂₉/S ₂₉ self-incompatible genotype of Brassica oleracea was screened with a probe carrying part of the catalytic domain of a Brassica S-receptor kinase (SRK)-like gene. Six positive phage clones with varying hybridisation intensities (K1 to K6) were purified and characterised. A 650–700 by region corresponding to the probe was excised from each clone and sequenced. DNA and predicted protein sequence comparisons based on a multiple alignment identified K5 as a pseudogene, whereas the others could encode functional proteins. K3 was found to have lost an intron from its genomic sequence. The six genes display different degrees of sequence similarity and form two distinct clusters in a dendrogram. The 98% similarity between K4 and K6, which extends across intron sequences, suggests that these might be very recently diverged alleles or daughters of a duplication. In addition, K2 showed a comparably high similarity to the probe. Clones K1, K3 and K5 cross-hybridised with an SLG ₂₉ cDNA probe, indicating the presence of upstream receptor domains homologous to the Brassica SLG gene. This suggests that the previously reported S sequence complexity may be ascribed to a large receptor kinase gene family.     

Structure and expression of AtS1, an Arabidopsis thaliana gene homologous to the S-locus related genes of Brassica

Summary Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequences were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.     

A new fermentation strategy for S-adenosylmethionine production in recombinant Pichia pastoris

A recombinant Pichia pastoris MutS expressing SAM2 gene of Saccharomyces cerevisiae was cultured for S-adenosylmethionine (SAM) accumulation. Effect of the amount of methanol added (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 6.0%, 10.0%, and 12.0%) and cell densities (9.57, 13.47, 21.74, 30.90, and 41.24 g/L dry cell weight (DCW)) on yield of SAM was found in flask cultivations. In flask experiments, maximal yield of SAM (1.29 g/L) was obtained at 2.0% methanol added and 30.90 g/L DCW which gave the maximal methanol consumption rate. Conjunct effect of amount of methanol added and cell density was found through Origin 7.0 (7.0 Microcal, USA). Scale up in 3.7 L bioreactor, 51% specific yield of SAM was enhanced at 0.6% methanol compared to that of 0.1% methanol. In fed-batches of different cell densities at 0.6% methanol, maximal yield of SAM was 8.66 g/L at 100 g/L DCW with 64% yield of SAM enhanced again. Methanol consumption rate at 100 g/L DCW was 4.81 mL/L h. Maintenance coefficient of 100 g/L DCW was lower than that of others significantly, although methanol consumption rate of 90 g/L DCW was higher (5.07 mL/L h) than that of 100 g/L DCW.     

表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定

为研制一种预防犬科动物狂犬病的新型疫苗,将含有狂犬病毒ERA株糖蛋白基因(Rabies glycoprotein,Rgp)表达盒的穿梭质粒pVAXΔE3Rgp中的Rgp表达盒克隆入犬2型腺病毒(Canine adenovirus type2,CAV2)骨架质粒pPoly2-CAV2中,获得重组质粒pPoly2-CAV2-ΔE3-Rgp,释放其基因组,转染MDCK细胞系,获得E3缺失区(Deletion of early protein3,ΔE3)含有Rgp表达盒的重组病毒CAV2-ΔE3-Rgp。该重组病毒能在MDCK细胞上产生典型的腺病毒细胞病变。通过酶切、PCR、基因测序,表明该重组病毒含有完整的Rgp表达盒。通过RT-PCR、Western blot等检测,表明该重组病毒能够表达Rgp抗原。用该重组病毒免疫犬,3次免疫后,可以诱导犬产生特异的抗CAV2HI抗体,其效价超过1∶256和抗狂犬病病毒(Rabies virus,RV)中和抗体,其效价超过0.50IU/mL。试验结果表明,获得的重组病毒免疫犬后,能够产生抗狂犬病毒和腺病毒的高效价保护性抗体,是一种有潜力的犬科动物狂犬病毒腺病毒二联疫苗候选株。     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

口蹄疫O型重组病毒的构建及其免疫原性分析

【目的】利用反向遗传操作技术,构建含O型口蹄疫病毒(food-and-mouth disease virus, FMDV) 3个拓扑型免疫优势结构蛋白基因的重组FMDV,评估其作为猪O型口蹄疫(food-and-mouth disease, FMD)疫苗候选株的潜力。【方法】通过基因合成,在FMD疫苗株O/HN/CHA/93 (古典中国拓扑型)的基因中嵌合流行株O/NXYCh/CHA/2018 (东南亚拓扑型) VP1结构蛋白的重组病毒骨架上,用O/TUR/5/2009疫苗株(中东-南亚拓扑型) VP1蛋白的G-H环基因替换其对等基因,构建含O型3个拓扑型FMDV结构蛋白基因的重组全长质粒,Not I线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救重组病毒。通过RT-PCR、序列测定、间接免疫荧光鉴定重组病毒;噬斑试验和一步生长曲线分析重组病毒的生物学特性。重组病毒制备疫苗免疫猪,用病毒中和试验分析其对当前流行的O型3个拓扑型FMDV的交叉反应性。【结果】成功拯救到含O型3个拓扑型FMDV结构蛋白基因的重组病毒,重组病毒与亲本病毒具有相似的生物学特性。亲本病毒和重组病毒制备的疫苗免疫猪,均能够对中东-南亚型(Middle East-South Asia, ME-SA)拓扑型和东南亚型(South-East Asia, SEA)拓扑型病毒株产生保护性平均中和抗体(>1.65log₁₀);均不能对古典中国型(Cathay)拓扑型流行株产生保护性平均中和抗体(<1.65log₁₀),但与亲本病毒相比,O/TUR/5/2009疫苗株G-H环基因的替换显著提高了对ME-SA和SEA拓扑型病毒株的交叉反应性(p<0.05)。【结论】本研究对未来FMD疫苗的设计具有重要的指导意义。     

SARS-CoV-2中和纳米抗体在里氏木霉中的重组表达北大核心CSCD

基于骆驼科动物单链抗体VHH结构域的纳米抗体具有分子量小、结构简单、溶解性好、稳定性强等多种优势,可通过吸入给药,在呼吸道病毒的防控中具有重要应用价值。里氏木霉是食品级的蛋白质生产宿主,其纤维素酶分泌量可达到80 g/L以上,有望用于药物蛋白的低成本生产。文中在密码子优化的基础上,使用组成型强启动子Pcdna1,实现了SARS-CoV-2中和纳米抗体Nb20在里氏木霉中的重组表达。将Nb20与里氏木霉纤维二糖水解酶CBHⅠ的N端片段融合表达,并在二者间引入胞内KEX2蛋白酶切位点,于葡萄糖培养基中摇瓶发酵48 h可生产出浓度为47.4 mg/L的Nb20蛋白。重组表达的纳米抗体能够与SARS-CoV-2刺突蛋白的受体结合区相结合,有望用于新型冠状病毒的中和。以上结果显示,里氏木霉在纳米抗体的重组表达中具有一定的应用潜力。     

Gas chromatographic-mass spectrometric determination of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol for study on human urinary excretion of ginsenosides after ingestion of ginseng preparations

An improved gas chromatographic-mass spectrometric method (GC-MS) with a fast solid-phase extraction on a newly introduced C₁₈ microcolumn, was applied to study the urinary excretion of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol glycosides in man after oral administration of ginseng preparations. Using panaxatriol as internal standard, 20(S)-protopanaxadiol and 20(S)-protopanaxatriol (the aglucones of ginesenosides) could be determined at a detection level of a few ng per ml urine by GC-MS with selected-ion monitoring after their release from glycosides which occur in urine. The extraction recovery of ginsenosides from urine was more than 80% and the intra-assay coefficient of variation was less than 5.0%. The results after intake of single doses of ginseng preparations demonstrated a linear relation between the amounts of ginsenosides consumed and the 20(S)-protopanaxatriol glycosides excreted in urine. About 1.2% of the dose was recovered in five days.