AcNPV增强子hr5增强HBsAg基因表达的研究

用形成包涵体（ＯＯＣ＋）并能利用人工合成启动序列和多角体ＸＩＶ启动子表达外源基因的转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３将多角体基因、乙型肝炎病毒表面抗原（ＨＢＳＡｇ）基因和苜蓿丫纹夜蛾核型多角体病毒（ＡｃＮＰＶ）的增强子ｈｒ５部分序列同时插入无包涵体的粉纹夜蛾核型多角体病毒ＴｎＮＰＶ－ＳＶＩ－Ｇ基因组中，得到两株高效表达ＨＢｓＡｇ基因又形成包涵体的重组病毒ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋．对重组病毒的酶切鉴定、ＤＮＡ斑点杂交和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证实，外源基因及其相应的启动子和增强子序列已正确插入病毒基因组中．插入顺序中，ｈｒ５增强子是插入ＨＢｓＡｇ基因下游，多角体基因与ＨＢｓＡｇ基因方向相反．１２５Ⅰ－固相放射免疫检测和Ｗｅｓｔｅｒｎｂｌｏｔ结果表明，ＨＢｓＡｇ基因在昆虫离体细胞中得到高效表达并保留了抗原活性．ＴｎＮＰＶ－ｓｈｒ２６－ＯＣＣ＋和ＴｎＮＰＶ－ｓｈｒ３５－ＯＣＣ＋表达的ＨＢｓＡ吕蛋白与没有插入增强子序列的重组病毒ＴｎＮＰＶ—ＨＢｓ８５－ＯＣＣ＋的比较，分别提高了４０％和４６％．     

Production of human neurotrophin-3 in baculovirus expression system

Human NT_3 gene was inserted into pPAK9 and the resultant plasmid was used to co_transfect sf21 cells with Bsu36 Ⅰ digested Bac6 genomic DNA. Recombinant baculovirus was isolated by plaque assay. Slot_hybridization, immunocytochemistry and serum_free culture of DRG demonstrated that NT_3 had been integrated into Bac6 DNA, expressed and secreted. The yield of purified hNT_3 was 200 μg from 200 mL cultural supernatant after passing CM Sepharose Fast Flow column, the concentration for its maximum bioactivity was 4 ng/mL.     

表达鸡传染性支气管炎病毒SD/97/01株S1蛋白的重组杆状病毒的构建

采用BAC－TO－BAC杆状病毒表达载体体系构建了表达鸡传染性支管炎病毒（IBV）呼吸型毒株SD／97／01S1蛋白的重组杆病病毒，含SD／97／01株S1基因原重组质粒p MDSD9701S1用BamHI和SalI双酶切后，回收的片段并克琶杆病病毒转座载体pFASTBACHTa中多角体基因启动子的下游，筛选出重组转座质粒pFASTSD9701S1U并转化大肠杆菌DH10BAC后,获得重组穿梭质粒rBacmidSD9701S1，用重组穿俊质粒DNA转染昆虫Sf9细胞，获得了含SD／97／01S1基因的重组杆状病毒rAcSD9701S1，重组病毒感染Sf9细胞后，用SDS－PAGE、Westernblot和IFA对细胞表达产物进行检测和分析。结果表明：构建的重组杆状病毒能够在昆虫细胞中表达SD／97／01的S1蛋白，该蛋白具有天然蛋白的抗原性。     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Cloning and expression of the vp39 gene of Bombyx mori nuclear polyhedrosis virus in E. coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction. Supported by the National Natural science Foundation of China and the Doctoral Foundation of Edn carto Committee Liu Deli: born in 1954, Doctoral Candidate from Huazhong Normal University To whom correspondence should be addressed: (027-7882712-2938)     

Cloning and expression of the vp39 gene ofBombyx mori nuclear polyhedrosis virus inE. coli

The nuclear capsid protein gene (vp39) ofBombyx mori nuclear polyhedrosis virus (BmNPV) was amplified successfully by PCR technique and inserted into pGEM 3zf(+). The 5′ and 3′ terminal area of the amplified vp39 gene were sequenced with silver-staining dideoxy method. Bmvp39 gene was sub-cloned into the expression vector pRSET-A, and transformed intoE. coli BL21. This gene was highly expressed by IPTG induction. SDS-PAGE analysis showed that the expressed protein is about 38 kd, and the expressed amount reached maxium in 4 h with IPTG induction. Supported by the National Natural science Foundation of China and the Doctoral Foundation of Edn carto Committee Liu Deli: born in 1954, Doctoral Candidate from Huazhong Normal University To whom correspondence should be addressed: (027-7882712-2938)     

马立克氏病病毒meq基因功能研究

从马立克氏病病毒（MDV）不同致病型毒株meq基因序列，meq基因产物及其细胞内表达特性和meq蛋白生物学功能的研究探讨马立克氏病病毒致瘤基因meq功能。完成了648A,CV1988/Rispens,814，广西地方毒株G2，N，0093，0095，0297，0304共9个MDV毒株meq基因的序列测定。MDV不同致病型毒株的meq基因序列相对比较保守，它们相互间核苷酸和氨基酸序列的同源性均很高；与所有7个致瘤的MDV毒株相比，在2个MDV-1弱毒疫苗CV1988/Rispens株和814株发现有二个特征性位点突变。此外，还在其ORF中首次发现含15个氨基酸残基（EELCAQLCSTPPPPI）的2个重复和含6个氨基酸残基（PPICTP）的4个重复，全分布在MEQ蛋白C-端的转录激活域内。MEQ蛋白的表达仅局限于感染细胞的核内，而且随感染时间增加。具有从核质向核仁和核膜转移趋向；Western Blotting和免疫沉淀试验证实重组杆状病毒感染细胞裂解物中有大小约为60kD的特异带，利用表达的MEQ蛋白产物免疫BALB/c小鼠，获得的杂交瘤细胞被克隆并与MDV感染的鸡胚成纤维细胞（CEF）做免疫荧光试验（FA），获得4株稳定产生抗MEQ蛋白单克隆抗体（McAb)的杂交瘤细胞，其中3G12E6单克隆抗体能够检测到MDV致瘤株感染的CEF及自然MD肿瘤细胞中表达的meq基因产物，而CV1988/Rispens感染的细胞则未检测到，发现细胞内表达的meq基因产物可明显促进MDVGA株对体外培养细胞的感染及增殖。研究结果表明，meq基因在感染细胞内的表达水平是MDV增殖和致病，致瘤的分子基因。     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

Sequencing of p35 gene fromHeliothis armigera polyhedrosis virus and its homologous comparison

Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino-acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification. Biography: WANG Ye-fu(1962-), male, Ph D.     

Enhanced hydrosen production by insertional inactivation of adhE gene in Klebsiella oxytoca HP1

Ethanol is the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1. Two moles of NAD（P）H are consumed to yield one mole of ethanol that may decrease bacterial hydrogen production. In this article the adhE gene that codes for acetaldehyde dehydrogenase was disrupted for the first time. A homologous recombination vector pTA-Str was constructed in which the adhE gene was disrupted by inserting an aminoglycoside-3＇-adenyltransferase （aadA） gene. As expected, the vector includes the insertion 5＇-adhE-aadA-adhE-3＇. The amplified DNA fragment 5＇-adhE-aadA-adhE-3＇ from pTA-Str was transformed into K. oxytoca HP1 and one recombinant was obtained. PCR analysis of the resulting genomic DNA indicated the appropriate deletion and insertion. Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant increased by 16.07% and ethanol concentration decreased by 77.47%, suggesting that inactivation of the adhE gene in K. oxytoca HP1 is a potential method for enhancing bacterial H2-production.     

短小芽孢杆菌(Bacillus pumilus)糖基转移酶基因的克隆、序列分析及表达

为获得具有催化活性的糖基转移酶纯酶,本研究以短小芽孢杆菌基因组DNA为模板,利用简并PCR技术扩增到基因(GT-A)全长序列.该序列全长1 287bp、编码423个氨基酸,分子量约为49.2KD.经序列分析,该基因属于糖基转移酶基因.根据GT-A基因开放阅读框序列设计引物,构建了原核表达重组质粒GTA-pet28a,并在大肠杆菌BL21(DE3)中成功诱导出了一个约50kD的融合蛋白.纯化后测定其糖基转移酶活性,与37℃相比,80℃的反应温度其活性能提高3.4倍左右.研究结果表明,该酶是一种具有应用潜力的嗜高温糖基转移酶.     

Production of human alpha-interferon in silkworm using a baculovirus vector

Microorganisms are generally used for mass production of foreign gene products, but multicellular organisms such as plants have been proposed as an economical alternative. The silkworm may be useful in this context as it can be cultured easily and at low cost. We have therefore developed a virus vector to introduce foreign genes, for example, the gene for human alpha-interferon (IFN-alpha), into silkworms. We used the baculovirus Bombyx mori nuclear polyhedrosis virus (BmNPV) which has a large (greater than 100 kilobases, kb) double-stranded circular DNA genome within its rod-shaped capsid. Baculoviruses have been used previously as vectors for expression of beta-interferon and beta-galactosidase in established cell lines. Although BmNPV has not been used previously as an expression vector, it has an advantage over the baculovirus Autographa californica NPV in that it has a narrower host range and will not grow in wild insect pests in the field. In the present study, the polyhedrin gene encoding the major inclusion body protein of BmNPV was identified by hybridization with complementary DNA and cloned in a plasmid. For insertion of foreign genes, we constructed a recombinant plasmid carrying a polylinker linked to the promoter of the polyhedrin gene, and inserted the IFN-alpha gene into this plasmid. The resulting plasmid and the BmNPV genomic DNA were co-transfected into BM-N cells, and stable recombinant viruses isolated by plaque assay on BM-N cells. The recombinant virus replicated in silkworm larvae, which synthesized as much as 5 X 10(7) units (approximately 50 micrograms) of interferon in their haemolymph.     

Cloning and characterization of a gene encoding cysteine proteases from senescent leaves of Gossypium hirsutum

A gene encoding a cysteine proteinase was isolated from senescent leave of cotton (Gossypium hirsutum) cv liaomian No. 9 by utilizing rapid amplification of cDNA end spolymerase chain reaction (RACE-PCR), and a set of consensus oligonucleotide primers was designed to anneal the conserved sequences of plant cysteine protease genes. The cDNA, which designated Ghcysp gene, contained 1368 bp terminating in a poly(A)^ trail, and included a putative 5‘(98 bp) and a 3‘(235 bp) non-coding region. The opening reading frame (ORF) encodes polypeptide 344 amino acids with the predicted molecular mass of 37.88 kD and theoretical pl of 4.80. A comparison of the deduced amino acid sequence with the sequence in the GenBank database has shown considerable sequence similarity to a novel family of plant cysteine proteases. This putative cotton Ghcysp protein shows from 67% to 82% identity to the other plants. All of them share catalytic triad of residues, which are highly conserved in three regions. Hydropaths analysis of the amino acid sequence shows that the Ghcysp is a potential membrane protein and localizes to the vacuole, which has a transmembrane helix between resides 7-25. A characteristic feature of Ghcysp is the presence of a putative vacuole-targeting signal peptide of 19-amino acid residues at the N-terminal region. The expression of Ghcysp gene was determined using northern blot analysis. The Ghcysp mRNA levels are high in development senescent leaf but below the limit of detection in senescent root, hypocotyl, faded flower, 6 d post anthesis ovule, and young leaf.     

可溶性人干细胞因子的原核表达和纯化

用Trizol提取人骨髓总RNA,通过RT-PCR扩增人干细胞因子DNA,克隆到pMD18-T载体中并测序．将其定向连入原核表达载体pET32a( ),获得了重组表达载体pET32a( )／hSCF,其cDNA长度为504bp,测序结果与已知序列吻合．然后在大肠杆菌BL21中经IPTG诱导表达,获得37kD的融合蛋白,约占菌体总蛋白量的30％．经Ni^2 -NTA树脂亲和层析柱纯化获得纯度大约90％的重组蛋白．     

利用Tn5转座子构建杆状病毒AcMNPV随机突变体的初步研究

以杆状病毒模式种AcMNPV为研究对象,应用基于Tn5转座子的随机转座的方法,构建杆状病毒突变体库将果蝇hsp70启动子后接绿色荧光蛋白基因后插入Tn5转座子,构建了可以在昆虫细胞中表达,易于跟踪的转座载体．利用体外转座系统将转座子随机插入AcMNPV基因组,并用转座反应液转染Sf21细胞,得到了表达绿色荧光蛋白的病毒突变体库进一步纯化了两株病毒B9F和Li6A,进行了转座子插入位点的分析,确定两株病毒中,转座子分别插入了94K基因和p10基因．该方法将为杆状病毒功能基因组研究提供重要的手段。     

马立克氏病病毒MEQ蛋白单克隆抗体的制备及应用研究

基因表达与病毒感染细胞及致瘤能力之间关系的阐明，将有助于我们对马立克氏病病毒（MDV）meq基因功能的了解，该研究利用通过杆状病毒载体在昆虫细胞系SF9上高度表达的MEQ蛋白免疫BALB/c小鼠，然后收获其免疫脾细胞与肿瘤细胞系SP2/0通过PEG于体外融合，获得的杂交瘤细胞被克隆并通过与MDV感染的鸡胚成纤维细胞（CEF）做免疫荧光试验（FA），进行其分泌抗MEQ单克隆抗体（McAb）能力的筛选，获得4株稳定产生抗MEQ蛋白McAb的杂交瘤细胞，其中3G12E6通过FA和免疫酶组化技术能够检测到MDV致瘤株感染的CEF及自然MD肿瘤细胞中表达的meq基因产物。研究的结果首次发现，MDV致瘤株GA、RB1B感染的CEF及自然MD肿瘤细胞中均有meq基因的表达，但在弱毒疫苗株CV1988感染的CEF和免疫鸡组织细胞中则未检测到MEQ，作者认为这是由于meq基因在致瘤株中表达水平高，而在非致瘤株中表达水平低所致，这可能是决定MDV毒力（virulence）或致瘤性（oncogenicity）的关键因素之一。     

Sequence analysis of mRNA polyadenylation signals of rice genes

Most eukaryotic mRNAs receive a poly (A) tail at their 3′-ends through a process involving the cleavage of pre-mRNA and the concomitant polymerization of adenosine residues to the cleaved RNA end[1,2]. As un- translated regions (UTRs) may contain importa…     

Detection of DNA methylation changes during seed germination in rapeseed （Brassica napus）

DNA methylation is a common yet important modi- fication of DNA in eukaryotic organisms. DNA methy- lation, especially methylation of cytosine (m5C), have both epigenetic and mutagenic effects on various cellu- lar activities such as differential gene exp…     

Apoptosis in suspension culture of tobacco cells induced by heat shock

The induction of apoptosis in suspension culture of tobacco cells by heat shock is reported for the first time. Heat treatment (48℃ for 4 h) of tobacco cells led to the appearance of typical hallmarks of apoptosis. It was demonstrated by DNA laddering analysis that the cells treated with heat shock at 48℃ for 4 h had a serious degradation of nuclear DNA into multi-nu-cleosomal sizes, suggesting that heat shock activated endogenous nuclease which led to DNA cleavage at the linkage sites between the nucleosomes, but ladders were very faint for DNA from 2 and 9 h heat-treated cells. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) detection also showed that most of these treated cells (48℃ for 4 h) displayed positive reactions, indicating a serious DNA 3'-OH cleavage in their nuclei. Moreover, some other cytological changes in apoptotic cells, such as cell shrinkage, chromatin aggregation, nucleus collapse, have also been observed by 4', 6'-diamidino-2-phenyl-indole (DAPI) staining.     

结核分枝杆菌H37Rv异柠檬酸裂解酶基因的克隆表达及活性

以结核分枝杆菌H₃₇Rv基因为模板, PCR反应扩增该菌株的异柠檬酸裂解酶基因（ICL）, 将其克隆入原核表达载体pET28b中, 并将pET28b I
CL转化入大肠杆菌BL21(DE3)中进行诱导表达. 结果表明, ICL蛋白的最佳诱导表达条件为: 温度20 ℃, IPTG终浓度为025 mmol/L, 诱导表达4 h, 在此条件下 ICL实现了高效表达, 以镍离子螯合型琼脂糖凝胶亲和层析柱纯化ICL蛋白, 纯化程度较高. 酶学性质鉴定表明, 实验获得了具有生物学活性的重组蛋白, 重组ICL的比活力为24 μmol/(mg·min).