应用PCR重组技术构建大肠杆菌分泌型表达载体

应用ＰＣＲ重组技术,对大肠杆菌分泌型表达载体ｐＩＮ－ⅢｏｍｐＡ进行改建,除去原载体的ＨｉｎｄⅢ位点,将信号肽序列末端两个密码子ＣＡＧ ＧＣＣ改为ＣＡＡ ＧＣＴ,从而引入一个新的ＨｉｎｄⅢ位点,并在其下游接上一段多克隆位点序列,改建后的载体可用于直接插入外源ＤＮＡ编码序更,表达产物在分泌过程中被切除信号肽而成为天然有活性的蛋白质,操作十分简便。     

E．coli分泌表达载体的构建和人表皮生长因子在E．coli中的分泌性表达

采用ＰＣＲ技术从Ｅ．ｃｏｌｉ基因组片段中克隆出碱性磷酸酯酶（ＰｈｏＡ）的启动子和信号肽序列．在ＰｈｏＡ启动予５＇端设计了ＥｃｏＲⅠ酶切位点,在信号肽编码序列３＇端设计了ＨｉｎｄⅢ酶切位点．将ＰＣＲ产物酶切后ＥｃｏＲⅠ－ＨｉｎｄⅢ片段克隆至ｐＢＲ３２２的ＥｃｏＲⅠ－ＨｉｎｄⅢ位点,组构出含有ＰｈｏＡ启动子和信号肽序列的分泌表达载体ｐＢＭ－Ｐｈｏ－１．之后将人表皮生长因子的成熟肽基因克隆至该载体,使之在Ｅ．ｃｏｌｉ中获得分泌表达,另采用ｐＩＮⅢ载体系统以分泌方式表达了人表皮生长因子。     

E.coli分泌表达载体的构建和人表皮生长因子在E.coli中的分泌…

采用ＰＣＲ技术从Ｅ．ｃｏｌｉ基因组片中克隆出碱性磷酸酯酶的启动子和信号肽序列,在ＰｈｏＡ启动子５端设计了ＥｃｏＲⅠ酶位点,在信号肽编码序列３端设计了ＨｉｎｄⅢ酶切位点,将ＰＣＲ产物酶切后ＥｃｏＲⅠ－ＨｉｎｄⅢ片段克隆至ｐＢＲ３２２的ＥｃｏＲⅠ－ＨｉｎｄⅢ倍点,组构出含有ＰｈｏＡ启动子和信号肽序列的分泌表达载体ｐＢＭ－Ｐｈｏ－,之后将人表皮生长因子的成熟肽基因克隆至该载体,使之有Ｅ．ｃｏｌｉ中获得分     

分泌载体pUS186的构建及地衣杆菌α—淀粉酶基因在枯草杆菌中的表…

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克枯草杆菌染色体的启动子和信号肽序列。将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ ３１酶切,Ｔ４ ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ｉ     

PARP酶抑制剂未引起整合的外源LacZ基因的丢失

使用聚ＡＤＰ核糖聚合酶（ＰＡＲＰ）ＮＡＤ位点抑制剂苯甲酰胺（ＢＡ）研究了降低ＰＡＲＰ酶活性对外源ＬａｃＺ基因整合稳定性的影响,利用ＤＮＡ体外重组技术将ＬａｃＺ基因全序列插入到真核表达载体载体ＰＳＶ２ｎｅｏ的ＨｉｎｄⅢ位点,构建了一个具有真核细胞ｎｅｏ基因筛选标记和ＬａｃＺ基因的真核表达重组体ＰＳＶ２ｎｅｏ－ｂｅｔａ－ｇａｌ将该重组体导入ＨｅＬａ细胞,经Ｇ４１８筛选获得了能稳定表达β－半乳糖苷酶的Ｈ     

分泌载体pUS186的构建及地衣杆菌α－淀粉酶基因在枯草杆菌中的表达和分泌

利用枯草杆菌的分泌系统构建分泌型表达载体表达和分泌外源基因产物具有重要的商业价值。我们用鸟枪法克隆了枯草杆菌染色体的启动子和信号肽序列,将克隆的序列连接到能在枯草杆菌中复制的质粒ｐＵＢ１８上,获得分泌型表达载体ｐＵＳ１８６。为了测试构建的载体ｐＵＳ１８６的功能,将地衣杆菌α－淀粉酶基因的缺失了启动子和信号肽序列的片段重组进该质粒,经过Ｂａｌ３１酶切,Ｔ４ＤＮＡ聚合酶补齐等处理,获得ｐＵＳＡ１８６Ⅱ及ｐＵＳＡ１８６Ⅰ系列质粒,将这些重组质粒转化枯草杆菌ＱＢ１１３０（ａｍｙ－）后都能向胞外分泌淀粉酶,酶活测定结果表明,基因表达水平比用原有的启动子高１－２倍,蛋白质分泌率在８４－９６％之间。     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

利用套叠PCR技术改造酵母表达载体pAO815的研究

利用套垒PCR技术将α-factor信号肽基因插入pA0815之EcoRⅠ位点,构建成分泌型表达载体pA0815α-A。在不改变原克隆位点EcoRⅠ的条件下,借助pA0815之第873位的HindⅢ位点,将pA0815AOX1启动子873-940片段连同α-factor信号肽基因序列（246bp）插入pA0815之HindⅢ/EcoRⅠ之间,构建成含α-factor信号肽基因的分泌型表达载体pA0815α-A。     

枯草杆菌启动子－信号肽序列的克隆及序列分析

利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序,结果表明,克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。     

双抗(抗病毒及抗虫)植物表达载体的构建及番茄的转化鉴定

将抗病毒的ＣＭＶ－ｃｐ 基因和抗虫的Ｂｔ－ｔｏｘｉｎ 基因依次插入到植物表达载体ｐＥ３ 的ＨｉｎｄⅢ和ＫｐｎⅠ位点,通过菌落原位杂交筛选和酶切鉴定,然后以土壤农杆菌ＧＶ３１１－ＳＥ介导转化番茄,胭脂碱检测,染色体ＤＮＡ 的点杂交及ＰＣＲ扩增证明ＣＭＶ－ｃｐ 基因和Ｂｔ－ｔｏｘｉｎ 基因已同时导入转化再生的番茄植株。ＲＮＡ 点杂交证明ＣＭＶ－ｃｐ 基因和Ｂｔ－ｔｏｘｉｎ 基因已在转基因番茄植株中同时获得表达。     

Expression of cDNA fragment encoding sperm membrane peptide inE. coli

A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P _R -P _L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.     

基因修饰对鸡新城疫病毒F48E9株HN基因DNA疫苗表达效力的影响

DNA疫苗的免疫效果与抗原基因的表达量和免疫原性有直接关系, 为了提高目的基因的表达量, 本研究对NDV F48E9株的HN基因进行了修饰, 对修饰前后HN基因表达水平进行了比较。利用分子生物学软件将NDV F48E9株的HN基因的密码子全部替换为鸡体内偏嗜性密码子, 同时在HN基因的5′端加上同样已替换密码子的禽流感病毒HA蛋白信号肽序列以期望提高目的蛋白在细胞中的表达。修饰后HN基因命名为SoptiHN, 剔除信号肽的HN基因命名为optiHN。将SoptiHN、optiHN和F48E9株的HN基因分别克隆到真核表达载体pVAX1和含有多个鸡体内最适免疫刺激序列CpG-ODN的载体pVAX1-CpG中, 将他们分别命名为pV-SoptiHN、pVC-SoptiHN、pV-optiHN、pVC-optiHN和pV-HN、 pVC-HN, 用这些质粒转染293T细胞, 48小时后间接免疫荧光和Western blotting检测细胞中瞬时表达的HN蛋白。结果显示, 与未经修饰的HN基因相比, 修饰后的HN基因体外瞬时表达水平明显提高, 并且密码子优化与添加信号肽序列这两种途径都可以提高HN基因的体外表达量。     

基因修饰对鸡新城疫病毒F48E9株HN基因DNA疫苗表达效力的影响

DNA疫苗的免疫效果与抗原基因的表达量和免疫原性有直接关系, 为了提高目的基因的表达量, 本研究对NDV F48E9株的HN基因进行了修饰, 对修饰前后HN基因表达水平进行了比较。利用分子生物学软件将NDV F48E9株的HN基因的密码子全部替换为鸡体内偏嗜性密码子, 同时在HN基因的5′端加上同样已替换密码子的禽流感病毒HA蛋白信号肽序列以期望提高目的蛋白在细胞中的表达。修饰后HN基因命名为SoptiHN, 剔除信号肽的HN基因命名为optiHN。将SoptiHN、optiHN和F48E9株的HN基因分别克隆到真核表达载体pVAX1和含有多个鸡体内最适免疫刺激序列CpG-ODN的载体pVAX1-CpG中, 将他们分别命名为pV-SoptiHN、pVC-SoptiHN、pV-optiHN、pVC-optiHN和pV-HN、 pVC-HN, 用这些质粒转染293T细胞, 48小时后间接免疫荧光和Western blotting检测细胞中瞬时表达的HN蛋白。结果显示, 与未经修饰的HN基因相比, 修饰后的HN基因体外瞬时表达水平明显提高, 并且密码子优化与添加信号肽序列这两种途径都可以提高HN基因的体外表达量。     

Plasmids pMB123 and pMB124--vectors for obtaining subfragments of DNA with random "sticky" ends

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.     

鸡肌球蛋白轻链激酶表达载体的构建

鸡肌球蛋白轻链激酶（ＭＬＣＫ）在调节平滑肌细胞收缩中具有重要作用．用ＰＣＲ产生构建所需的限制性内切酶ＳａｌⅠ位点,将鸡ＭＬＣＫｃＤＮＡ插入质粒ｐＢＫｒｓｖ中,构建成ｐＢＫｒｓｖ－ＭＬＣＫ,并通过酶切图谱、ＳａｌⅠ位点序列分析得到证实．重组质粒在大肠杆菌中获得表达．     

Soluble expression of recombinant olive flounder hepcidin I using a novel secretion enhancer

Expression of olive flounder hepcidin I (HepI) fused with truncated OmpA signal peptides (OmpASP(tr)) as directional signals does not produce soluble fusion proteins. However, by inserting amino acid segments (xxx) varying in pI and hydrophobicity/hydrophilicity into a leader sequence containing a truncated OmpASP (OmpASP(tr)) and a factor Xa cleavage site (Xa) [OmpASP(tr)|(xxx)|Xa], we were able in some cases to express soluble recombinant HepI. Soluble expression of the recombinant protein strongly correlated with (xxx) insertions of high pI and hydrophilicity. Therefore, we modified the OmpASP(tr)|(xxx)|Xa sequence by inserting Arg and Lys into (xxx) to increase the hydrophilicity of the signal peptide region. These modifications enhanced the expression of soluble recombinant HepI. Hydropathic profile analysis of the OmpASP(tr)|(xxx)|Xa| HepI fusion proteins revealed that the transmembrane-like domains derived from the OmpASP(tr)|(xxx)|Xa sequence were larger than the internal positively charged domain native to HepI. It should therefore be possible to overcome the obstacle of internal positively charged domains to obtain soluble expression of recombinant proteins by monitoring the hydrophilicity and hydropathic profile of the signal peptide region using a computer program.     

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.     

Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes

The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process. Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences. Moreover, the synthetic sequences cloned are often altered versions of those originally designed. We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame. The test is the reversal of the beta-galactosidase alpha-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors. We begin with a modified vector defective in alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation. The alpha-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment. The alpha-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites. Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy. We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.