牛疱疹病毒Ⅰ型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活作用

由含有ＢＨＢＶ－１（ＢｏｖｉｎｅＨｅｒｐｅｓＶｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＢＩＣＰＯ（ＢＨＶ－１ＩｎｆｅｃｔｅｄＣｅｌｌＰｒｏｔｅｉｎＯ）的ＤＮＡ序列至表达载体ｐＳＶＫ３,构建质粒ｐＳＶ２．９。将该质粒与ｐＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶＬＴＲ启动子控制下的荧光素酶基因的表达。根据ｐＳＶ２．９与含有ＢＩＶＬＴＲ不同区段缺失的质粒ｐＤ－３１９－Ｌｕｃ、ｐＤ－１１５－Ｌｕｃ、ｐＤ－５２－Ｌｕｃ共转染小牛肺细胞的实验结果,推测ＢＩＶＬＴＲ－３１９位上游区的ＤＮＡ序列影响ＢＩＣＰＯ基因产物对ＢＩＶＬＴＲ表达的激活作用。     

大鼠催乳素基因真核细胞可表达性质粒的构建及应用研究

７３５ｂｐ的ＰＲＬｃＤＮＡ片段从质粒ＰＲＬ－ＳＰ６５＃１中回收后,用粘性末端连接法将其重组到真核表达载体ｐｃＤＮＡ３上,筛选出正向连接重组体ｐｃＤＮＡ３－ＰＲＬＳ和反向连接重组体ｐｃＤＮＡ３－ＰＲＬＡＳ。将重组体ｐｃＤＮＡ３－ＰＲＬｓ和空载体ｐｃＤＮＡ３分别转入ＮＩＨ３Ｔ３细胞系,用Ｇ４１８筛选出阳性细胞后与未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２的情况下,用原位杂交的方法,分别用ＰＲＬｃＤＮＡ探针和原癌基因ｃ－Ｈ－ｒａｓｃＤＮＡ探针进行检测,未转染的ＮＩＨ３Ｔ３细胞在加Ｅ２和不加Ｅ２时都几乎无催乳素基因的表达,同样,转入空载体的ＮＩＨ３Ｔ３细胞也无ＰＲＬ的表达,而转入重组体ｐｃＤＮＡ３－ＰＲＬＳ的ＮＩＨ３Ｔ３细胞则有大量的ＰＲＬ基因的表达,与对照组相比有显著差异（Ｐ＜０．０１）。正常和转入空载体的ＮＩＨ３Ｔ３细胞有一定程度的原癌基因ｃ－Ｈ－ｒａｓ的表达,当分别加入Ｅ２和转入重组体ｐｃＤＮＡ３－ＰＲＬＳ后,ＮＩＨ３Ｔ３细胞中的ｃ－Ｈ－ｒａｓ基因表达水平都显著升高（Ｐ＜０．０５）。     

小鼠4.5S RNA逆转座子对Luc基因表达的影响

利用ＲＴ－ＰＣＲ方法,从小鼠肝脏组织总ＲＮＡ中扩增出４．５ＳＲＮＡ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,酶切鉴定并测序。然后将该序列插入以Ｌｕｃ基因作为报道基因的表达载体ｐＳＶｌｕｃ２０的ＰｖｕⅡ位点,构建了含４．２ＳＲＮＡ逆转座子的表达载体ｐＳＶｌｕｃ２０－４．５Ｓ。脂质转染法将表达载体导入小鼠骨髓瘤细胞ＮＳ－１、ＳＰ２／０和人乳腺癌细胞Ｂｃａ６１。结果表明,小鼠４．５ＳＲＮ     

牛疱疹病毒I型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活…

由含有ＢＨＶ－１（Ｂｏｖｉｎｅ Ｈｅｒｐｅｓ Ｖｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＩＣＰＯ（ＢＨＶ－１Ｉｎｆｅｃｔｅｄ Ｃｅｌｌ Ｐｒｏｔｅｉｎ Ｏ）的ＤＮＡ序列至表达载体ｐＳＶＤ３,构建质粒ｐＳＶ２．９。将该质粒与ＰＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶ ＬＴＲ启动子控制下的荧光素酶基因的表达。根据ＰＳＶ２．９与含有Ｂ     

HCV非结构蛋白(NS2/NS3)基因表达载体的构建及其一级结构分析

应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术,从ＨＣＶ感染者血清中扩增编码ＨＣＶ病毒蛋白酶的ＮＳ２－ＮＳ３ｃＤＮＡ片段,在其５′和３′端分别引入ＥｃｏＲⅠ和ＸｂａⅠ限制性内切酶位点,定向克隆至真核表达载体ｐｃＤＮＡ３,构建重组载体ｐｃＤＮＡ－ＮＳ２３,重组表达载体经限制性内切酶消化鉴定．用ＳＰ６和Ｔ７通用引物对目的基因片断进行序列分析．序列同源性分析结果表明,与ＨＣＶ－Ｊ、ＨＣ－Ｃ２有高度的同源性,与ＨＣＶ－１、ＨＣＶ－Ｊ６、ＨＣＶ－Ｊ８同源性差,提示所克隆的基因属ＨＣＶⅡ型．该区内重要的功能位点如Ｚｎ２＋依赖性金属蛋白酶催化中心、丝氨酸蛋白酶催化中心等均高度保守     

Egr-1基因调控序列连接OSM cDNA表达质粒的构建及其在黑色素瘤细胞中的表达

ＯＳＭ是一种对黑色素瘤细胞显示抑制作用的细胞因子．为进行ＯＳＭ针对黑色素瘤的基因－放射治疗研究,构建了小鼠Ｅｇｒ－１基因调控序列引导入ＯＳＭｃＤＮＡ真核表达质粒（ｐＥＯ）,ｐＥＯ质粒转染小鼠Ｂ－１６黑色素瘤细胞,经Ｇ４１８和抗人ＯＳＭ抗体的筛选,获得了稳定表达ＯＳＭ的克隆细胞（ｐＥＯ－１细胞）,ＯＳＭ表达量可达５．９７ｎｇ每１０５细胞天,分子量为３２ｋＤ．ｐＥＯ－１细胞用一定浓度Ｈ２Ｏ２处理后ＯＳＭ表达量可提高６２％,表明ｐＥＯ重组质粒可在氧自由基的刺激作用下增强ＯＳＭ表达     

小鼠白细胞介素12双顺反子真核表达载体的构建及其在COS-7细胞中的表达

克隆小鼠白细胞介素１２（ＩＬ－１２）ｐ４０及ｐ３５ｃＤＮＡ,并构建同时含ｍＩＬ－１２ｐ４０和ｐ３５ｃＤＮＡ的双顺反子真核表达载体及其在哺乳动物细胞中的表达．白细胞介素１２是由巨噬细胞,树突状细胞等抗原提呈细胞产生的一种异二聚体细胞因子,对机体的细胞免疫功能起着重要的调节作用．利用脂多糖（１００ｐｇ／ｍｌ）和小鼠重组干扰素（ＩＦＮ－γ５００Ｕ／ｍｌ）体外联合刺激小鼠腹腔巨噬细胞,从中提取总ＲＮＡ,经ＲＴ－ＰＣＲ扩增出含信号肽的小鼠白细胞介素１２（ｍＩＬ－１２）ｐ４０及ｐ３５全长ｃＤ－ＮＡ．ＰＣＲ产物经酶切后,分别克隆至ｐＢｌｕｅｓｃｒｉｐｔⅡＳＫ载体中,序列测定结果与文献报道序列一致．然后利用脊髓灰质炎（Ｐｏｌｉｏ）病毒内核糖体进入位点（ＩＲＥＳ）连接ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ,亚克隆至ｐｃＤＮＡ３载体中,构建成含ｍＩＬ－１２ｐ４０及ｐ３５ｃＤＮＡ双顺反子真核表达载体,即ｐｃＤＮＡ３／ｍＩＬ－１２,ｐ４０及ｐ３５ｃＤＮＡ同时受ｐｃＤＮＡ３中ｈＣＭＶ启动子驱动,将ｐ４０及ｐ３５转录至同一ｍＲ－ＮＡ上．通过ＬｉｐｏｆｅｃｔＡＭＩＮＥ将ｐｃＤＮＡ３／ｍＩＬ－１２转染ＣＯＳ－７细胞,７２ｈ收集培养上清,测定ｍ     

转译优化对EPO基因在COS7细胞中表达的影响

利用ＣＯＳ７细胞暂时表达系统,研究转译起始序列对ＥＰＯ－ｃＤＮＡ表达的影响。通过ＤＮＡ重组技术,构建了原ＥＰＯ－ｃＤＮＡ表达载体ｐＣＳＶ－ＥＰＯ（１）,其转译起始序列为５＇ＡＡＴＴＣＡＴＧＧ３＇。同时通过定点突变技术,将起始序列改变成５＇ＣＣＡＣＣＡＴＧＧ３＇,而构建了另一表达载体ＰＣＳＶ－ＥＰＯ（２）。后经序列分析证明无误后和前均通过ＤＥＡＥ－ｄｅｘｔｒａｎ法转染ＣＯＳ７细胞上清,测定结果为     

人促红细胞生成素基因组基因和cDNA的克隆及其在COS－7细胞中的表达

利用ＰＣＲ技术,从正常人胎肝染色体ＤＮＡ中克隆到长度为１５７２ｂｐ的人促红细胞生成素（ＥＰＯ）基因组基因片段,它包含除第一个外显子和第一个内含子外所有外显子及内含子。再人工合成１３ｂｐ外显子１的编码区,并与１５７２ｂｐ片段拼接,从而得到除第一个内含子的人促红细胞生成素基因组基因。将克隆得到的ＥＰＯ基因插入载体ｐＳＶ２－ｄｈｆｒ得到ｐＳＶ２－ＥＰＯ表达载体,转染ＣＯＳ－７细胞后获得高效表达。利用自行研制的小鼠抗人ＥＰＯ单抗及兔抗人ＥＰＯ多抗,对表达产物进行ＥＬＩＳＡ定量测定,细胞分泌ＥＰＯ量高达２５１±７Ｕ／ｍｌ．Ｋｒｙｓｔａｌ法测得体外生物活性２４１．５±６．５Ｕ／ｍｌ．用ＥＰＯ单抗免疫沉淀结合ＳＤＳ－ＰＡＧＥ对转染细胞的表达产物做了进一步鉴定,清晰地看到了ＥＰＯ条带。从高效表达ＥＰＯ的转染细胞中分离纯化ｍＲＮＡ,用ＲＴ－ＰＣＲ方法扩增并克隆到ＥＰＯ的ｃＤＮＡ,这为ＥＰＯ在其它系统中的表达及ＥＰＯ的功能与结构的研究打下了基础。     

用T—A载体克隆技术构建丙肝病毒C＋E1区真核表达质粒pSVL—HC…

用ＢａｍＨＩ和ＨｉｎｄⅢ将丙肝病毒Ｃ＋Ｅ１ＤＮＡ片段从其克隆载体ｐＧＥＭ３ｚｆ－ＨＣＶ／Ｃ＋Ｅ１上切下,经Ｔｘｑ酶补齐３‘末端后插入到载体ｐＳＶＬ－Ｔ中,构建成丙肝病毒Ｃ＋Ｅ１真核表达载体ｐＳＶＬ－ＨＣＶ／Ｃ＋Ｅ１。     

Interaction of ribosomal proteins S5, S6, S11, S12, S18 and S21 with 16 S rRNA

We have examined the effects of assembly of ribosomal proteins S5, S6, S11, S12, S18 and S21 on the reactivities of residues in 16 S rRNA towards chemical probes. The results show that S6, S18 and S11 interact with the 690-720 and 790 loop regions of 16 S rRNA in a highly co-operative manner, that is consistent with the previously defined assembly map relationships among these proteins. The results also indicate that these proteins, one of which (S18) has previously been implicated as a component of the ribosomal P-site, interact with residues near some of the recently defined P-site (class II tRNA protection) nucleotides in 16 S rRNA. In addition, assembly of protein S12 has been found to result in the protection of residues in both the 530 stem/loop and the 900 stem regions; the latter group is closely juxtaposed to a segment of 16 S rRNA recently shown to be protected from chemical probes by streptomycin. Interestingly, both S5 and S12 appear to protect, to differing degrees, a well-defined set of residues in the 900 stem/loop and 5'-terminal regions. These observations are discussed in terms of the effects of S5 and S12 on streptomycin binding, and in terms of the class III tRNA protection found in the 900 stem of 16 S rRNA. Altogether these results show that many of the small subunit proteins, which have previously been shown to be functionally important, appear to be associated with functionally implicated segments of 16 S rRNA.     

Expression and purification of human ribosomal proteins S3, S5, S10, S19, and S26

The cDNAs for the human ribosomal proteins S3, S5, S10, S19, and S26 were introduced into a pET-15b vector and recombinant proteins containing an N-(His)(6)-fusion tag were expressed in high yields. To resolve the problem of frameshift during expression of S26 caused by the presence of tandem arginine codons in its mRNA that are rare in Escherichia coli, we substituted the rare AGA codon with the more frequent arginine codon (CGC) using a primer with this mutation for PCR amplification of S26 cDNA. All proteins were expressed mainly in the form of inclusion bodies and purified to homogeneity by metal affinity chromatography in one step (except for S3). Expression of the full-length S3 was accompanied by the formation of a low molecular weight polypeptide that was co-purified with S3 by metal affinity chromatography. Complete purification of S3 required an additional gel-filtration step. The proteins were refolded by stepwise dialysis. Both identity and purity of the proteins were confirmed by 2D PAGE. The proteins obtained could be used in a wide range of applications in biophysics, biochemistry, and molecular biology.     

Positions of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit of Escherichia coli

Neutron scattering distance data are presented for 33 protein pairs in the 30 S ribosomal subunit from Escherichia coli, along with the methods used for measuring distances between its exchangeable components. When combined with prior data, these new results permit the positioning of S2, S13, S16, S17, S19 and S21 in the 30 S ribosomal subunit, completing the mapping of its proteins by neutron scattering. Comparisons with other data suggest that the neutron map is a reliable guide to the quaternary structure of the 30 S subunit.     

Interaction of proteins S16, S17 and S20 with 16 S ribosomal RNA

We have used rapid chemical probing methods to examine the effect of assembly of ribosomal proteins S16, S17 and S20 on the reactivity of individual residues of 16 S rRNA. Protein S17 strongly protects a compact region of the RNA between positions 245 and 281, a site previously assigned to binding of S20. Protein S20 also protects many of these same positions, albeit more weakly than S17. Strong S20-dependent protections are seen elsewhere in the 5' domain, most notably at positions 108, and in the 160-200 and 330 loop regions. Enenpectedly, S20 also causes protection of several bases in the 1430-1450 region, in the 3' minor domain. In the presence of the primary binding proteins S4, S8 and S20, we observe a variety of effects that result from assembly of the secondary binding protein S16. Most strongly protected are nucleotides around positions 50, 120, 300 to 330 and 360 in the 5' domain, and positions 606 to 630 in the central domain. In addition, numerous nucleotides in the 5' and central domains exhibit enhanced reactivity in response to S16. Interestingly, the strength of the S20-dependent effects in the 1430-1450 region is attenuated in the presence of S4 + S8 + S20, and restored in the presence of S4 + S8 + S20 + S16. Finally, the previously observed rearrangement of the 300 region stem-loop that occurs during assembly is shown to be an S16-dependent event. We discuss these findings with respect to assignment of RNA binding sites for these proteins, and in regard to the co-operativity of ribosome assembly.     

Immunoprecipitation of 70S, 50S and 30S ribosomes ofEscherichia coli

Antibodies were raised in rabbits against 70S ribosomes, 50S and 30S ribosomal subunits individually. Purified immunoglobulins from the antiserum against each of the above ribosomal entities were tested for their capabilities of precipitating 70S, 50S and 30S ribosomes. The observations revealed the following: (i) The antiserum (IgG) raised against 70S ribosomes precipitates 70S ribosomes completely, while partial precipitation is seen with the subunits, the extent of precipitation being more with the 50S subunits than with 30S subunits; addition of 50S subunits to the 30S subunits facilitates the precipitation of 30S subunits by the antibody against 70S ribosomes. (ii) Antiserum against 50S subunits has the ability to immunoprecipitate both 50S and 70S ribosomes to an equal extent. (iii) Antiserum against 30S subunits also has the property of precipitating both 30S and 70S ribosomes. The differences in the structural organisation of the two subunits may account for the differences in their immunoprecipitability.     

Structural and functional studies on Ribonuclease S, retro S and retro-inverso S peptides

Ribonuclease S peptide and S protein offer a unique complementation system to understand the finer features of molecular recognition. In the present study the S peptide (1-16), and its retro and retro-inverso analogs have been analyzed for their structural and biological attributes. RPHPLC, CD, and NMR analyses have revealed that the physicochemical and conformational properties of the S peptide are distinct from those of its retro and retro-inverso analogs. On the functional side, while the S peptide complemented the S protein to give RNase activity, was recognized by anti-S peptide antibodies and induced T cell proliferation, neither the retro nor the retro-inverso S peptides could do so.     

Immunochemical accessibility of ribosomal protein S4 in the 30 S ribosome. The interaction of S4 with S5 and S12

The reactivity of protein S4-specific antibody preparations with 30 S ribosomal subunits and intermediates of in vitro subunit reconstitution has been characterized using a quantitative antibody binding assay. Anti-S4 antibody preparations did not react with native 30 S ribosomal subunits; however, they did react with various subunit assembly intermediates that lacked proteins S5 and S12. The inclusion of proteins S5 and S12 in reconstituted particles resulted in a large decrease in anti-S4 reactivity, and it was concluded that proteins S5 and S12 are primarily responsible for the masking of S4 antigenic determinants in the 30 S subunit. The effect of S5 and S12 on S4 accessibility is consistent with data from a variety of other approaches, suggesting that these proteins form a structural and functional domain in the small ribosomal subunit.     

Positions of proteins S6, S11 and S15 in the 30 S ribosomal subunit of Escherichia coli

A map of the positions of 12 of the 21 proteins of the 30 S ribosomal subunit of Escherichia coli (S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12 and S15), based on neutron scattering, is presented and discussed. Estimates for the radii of gyration of these proteins in situ are also obtained. It appears that many ribosomal proteins have compact configurations in the particle.     

Positions of proteins S14, S18 and S20 in the 30 S ribosomal subunit of Escherichia coli

A map of the 30 S ribosomal subunit is presented giving the positions of 15 of its 21 proteins. The components located in the map are S1, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S14, S15, S18 and S20.