半衰期延长获得PAI－1抗性的t—PA突变体的构建、表达及特性分析

用基因重组及定位突变技术成功地构建了t·PA的Kl区缺失突变体t—PAdelKl、PAI-1结合位点缺失突变体t—PA del(296—302)及两者的组合突变体t—PA del(Kl·296—302),并在COS－7细胞中实现三者的暂时性表达,在cHO细胞中实现了t·PA deI(K1．296—302)的稳定性表达．对表达产物的生物学特性分析表明,t—PA del(296—302)及t-PA del(K1,296—302)获得了Pal－1抗性．因时t-PA del(K1．296—302)在大鼠体内的半衰期延长约5倍,而与纤维蛋白的亲和力只略有下降。因此,此组合突变体有可能成为优于野生t—PA的新型溶栓剂候选株．     

蚓激酶基因的人工合成及山羊乳腺表达

为了实现蚓激酶在真核细胞的高效稳定表达 ,人工合成了全长 849bp ,不含罕用密码子的蚓激酶F-Ⅲ-1cDNA序列 ,并以其为目的基因 ,构建以山羊β-酪蛋白启动子为上游调控序列的乳腺组织特异性表达载体pBLK和重组逆转录病毒表达载体pLN-bCP-LK。质粒pBLK 40 0 μg直接注入稳定泌乳期奶山羊乳腺 ,以纤维蛋白平板溶圈法 (FAPA)检测奶样纤溶活性。结果显示 ,注射后 3～ 60h ,奶样有明显纤溶活性 ,其中 6～ 9h活性最高。脂质体介导质粒pLN-bCP-LK转染PA317细胞系 ,5 0 0mg/LG418筛选后获得 4株高产毒细胞系 ,产毒滴度达 1× 1 0 ⁴～ 1× 1 0 ⁵CFU/ml水平。产毒细胞上清 1 0ml直接注入临产前两周的山羊乳腺 ,隔日以等量再注 1次 ,产羔后其奶样即有明显纤溶活性 ,注后第 45日纤溶活性仍无明显降低。     

人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。     

几种乙型肝炎病毒DNA重组质粒的改建及其在哺乳动物细胞高效表达乙型肝炎表面抗原

将Bgl Ⅱ不完全水解pTHBV一1质粒产生的两种乙型肝炎病毒(HBV)DNA片段插入pSV2-dhft质粒的Bglll切口,构造成{种重组质粒,其结构特点是：1都含有SV40的DNA复制起点和早期启动子;2都含有二氢叶酸还原酶基因(dhfr) 3·{种重组质粒是在dhfr。基因3’末端分别插入两种大小不同,方向相反的HBV’DNA片段而成。使用这4种质粒DNA分别和tk质粒DNA共转化Ltr细胞,在HAT培养基选择压力下4种质粒都得到有效表达HBsAg。通过换用氨甲喋呤并逐渐增加其药量,从氨甲喋呤抗性细胞中获得高效表达HBsAg的B2和B16细胞系。据固相放射免疫检测结果计算,B16细胞系的HBxAF分泌量可达12．5pg／10’细胞／天．细胞传代4个月表达稳定。这些细胞在HBsAg诊断试剂和疫苗研制中具有潜在的应用价值。     

鼠OAZ1基因的真核表达质粒的构建及其在COS7细胞中的表达

构建鼠OAZ1基因真核表达质粒,观察OAZ1-GFP融合蛋白在绿猴肾细胞COS7中的表达。利用RT-PCR的方法获得鼠OAZ1基因,再利用重叠延伸PCR缺失掉OAZ1序列中的205位碱基T,由此获得无需阅读框移码即可编码全长OAZ1的突变基因。将此突变基因克隆入真核表达载体pEGFP-N1获得重组质粒pEGFP-N1-OAZ1-T。将此质粒瞬时转染COS7后,采用RT-PCR,Western blotting,免疫荧光技术观察检测目的基因的表达情况。结果显示,酶切和测序证明质粒pEG-FP-N1-OAZ1-T构建正确,转染COS7细胞后,OAZ1-GFP融合蛋白能在细胞中高效表达。成功构建了pEGFP-N1-OAZ1-T真核表达质粒,在COS7细胞内,该质粒能成功指导OAZ1-GFP融合蛋白合成。     

copGFP和PuroR双标记基因慢病毒融合表达载体的构建及应用*

目的: 构建具有绿色荧光蛋白(copGFP)和嘌呤霉素抗性基因(PuroR)融合表达双筛选标记的慢病毒过表达载体,检测其嘌呤霉素抗性和绿色荧光蛋白表达的特性。方法: 从pCDH-CMV-MCS-copGFP载体中扩增copGFP编码区DNA序列,从pLKO.1载体中扩增Puro^R编码区DNA序列,运用重组PCR方法,扩增copGFP与Puro^R基因融合编码序列并克隆至经BamH Ⅰ+Sal Ⅰ双酶切的pCDH-CMV-MCS-copGFP载体片段中,构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体;载体的融合标签序列进行测序确证;将该载体用辅助包装质粒PLP1、PLP2、VSVG在293T细胞中包装成慢病毒后感染肝癌细胞MHCC97H,检测感染细胞对嘌呤霉素的抵抗作用以及绿色荧光蛋白的表达情况;为验证该载体表达外源目的基因的有效性,将Sp1编码区DNA序列插入该载体中包装成慢病毒,用对照及表达Sp1的慢病毒感染肝癌细胞MHCC97H,感染细胞经1 mg/ml嘌呤霉素筛选7 d后获得稳定感染细胞株,提取稳定感染细胞的总RNA及总蛋白,分别运用RT-qPCR和Western blot方法检测Sp1在对照及表达Sp1的慢病毒感染的肝癌MHCC97H细胞中的mRNA和蛋白表达水平的差异。结果: 成功构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体;该载体与辅助质粒包装出的慢病毒感染肝癌细胞后,感染细胞同时具有嘌呤霉素抗性和表达绿色荧光蛋白特性;将Sp1编码序列插入该载体,包装慢病毒并肝癌细胞,Sp1 的mRNA水平对照细胞相比分别升高3.3倍,蛋白水平升高2.2倍(P＜0.01)。 结论: 成功构建含copGFP和Puro^R融合表达双筛选标记的慢病毒过表达载体,该载体编码的融合双标记基因具有嘌呤霉素抗性和绿色荧光蛋白表达的特性,可高水平表达长片段的目的基因。     

LRP16对乳腺癌MCF-7细胞增殖的影响

用Northern印迹方法检测雌二醇 (17β E2 )对LRP16mRNA表达的时间及剂量依赖性调控作用 .构建LRP16基因启动子序列调控的萤光素酶报告子 (pS0 ) ,并与雌激素受体α和 β(ERα和ERβ)表达载体共转染COS 7和MCF 7细胞后测定萤光素酶活性 .将LRP16基因的表达载体转染MCF 7细胞 ,测定过表达LRP16对细胞的生长特性的影响 .17β E2 使MCF 7细胞中LRP16mRNA表达水平增加 ,增加幅度未显示出 17β E2 培养时间和剂量的依赖性 .pS0 与ERα表达载体共转染细胞的相对萤光素酶活性较非共转染组 (对照组 )及pS0 ERβ表载体共转染组升高 5～ 10倍 .LRP16基因过表达促进MCF 7细胞的增殖 .研究表明 ,雌激素可能通过ERα上调乳腺癌MCF 7细胞LRP16基因的表达并促进细胞增殖     

组织型纤溶酶原激活剂(t-PA)全长cDNA序列的克隆及表达

在先前获得t—PA部分编码序列的基础上,用化学合成法合成了缺少的5′端部分序列,并经多次重组获得了含有t—PA全长cDNA的重组表达质粒pMG-601。经序列分析表明该cDNA序列是正确的,将其导入暂时表达系统COS-7细胞中,能产生有特异性的t—PA产物。将其导入CHO细胞中,经MTX加压扩增,获得产t-PA的细胞株,其表达水平约为400～500IU/(10~6cells·24hr)。     

ut-PA抗PAI-1突变体的构建及在sf-9细胞中的表达

为使嵌合分子 ut- PA获得抗 PAI- 1抑制作用的性质 ,将删除了编码 u- PA中 R1 78- R1 79-H1 80 - R1 81的 1 2个核苷酸的 u- PA c DNA[u- PA( 1 ) ]的 Bam H - Eco R 部分酶切片段 ,克隆到含嵌合蛋白 ut- PA基因的转移载体 p VL 1 392 - ut- PA的相应位点中 ,构建了一个含有新的嵌合蛋白基因 ut- PA( 1 )的转移表达载体 p VL1 392 - ut- PA( 1 ) .在昆虫病毒表达系统 sf- 9细胞中表达该嵌合蛋白基因 ,表达上清具有纤溶性 ,用血纤维蛋白平板法和 S2 4 44 显色底物法分别测得活力为 2 4 8IU/ml和 380 IU/ml     

人尿激酶原cDNA在中国仓鼠卵巢细胞(CH0)中高效表达研究

通过构建高效表达载体,改进转染方法,与二氢叶酸还原酶(dhfr)基因共扩增等手段在CH0细胞内高效表达了人尿激酶原(Pr0-UK)cDNA。首先将pro—UK cDNA插入到sR α启动子的下游,构建成表达质粒pMGl0102,在cos－7细胞内进行暂时性表达,结果表明此启动子的表达水平比SV40早期启动子高约5倍。然后将质粒pMG10102和pSV2－dhfr线性化后用磅酸钙共沉淀法转染CH0－dhfr-细胞,经一系列筛选后获得20个能表达pro—UK的细胞克隆,纤维蛋白溶解平板法(FAPA)测定表达水平为12．5—100IU／10⁶cells／d．再经MTX加压共扩增,得到9株高表达细胞系,其中最高的表达水平达到400—500Iu／10‘cells／d。2—3个月连续传代,表达水平未下降,表明细胞株是稳定的。Western Blot分析证明细胞分泌的重组pro－UK具有与天然pro—UK相同的分子量,而且培养液中不加蛋白酶抑制剂时,分泌的重组UK大部分为单链(60％以上)。     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

Reinforcing apoptosis-resistance of COS and myeloma cells by transfecting with bcl-2 gene

COS, myeloma and HeLa cells, which are commonly used for protein production by cell culture, were transfected with human bcl-2 gene encoded on the shuttle vector BCMGS. Expression of human bcl-2 improved survival of cells remarkably, mildly, or negligibly for COS, myeloma, and HeLa, respectively. Four clones were obtained from the human bcl-2 expressing cell population of COS cells. They expressed human bcl-2 almost at the same level. The viable cell numbers were 6, 2.5, 2.5, and 0.8 times as many for the clones #8, #5, #6, and #7, respectively, as for the control COS cells, when they were cultured at low (0.2%) serum concentration for 9 days. The bcl-2 overexpressing COS cells showed morphology different from that of the control COS cells in serum limited condition. When transfected with mouse lambda protein gene carried by an SV40-derived vector, clone #8 of the bcl-2 transfected COS cells continued the transient expression of lambda protein longer than the control COS cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.     

Stable expression of human tissue-type plasminogen activator regulated by beta-actin promoter in three human cell lines: HeLa, WI-38 VA13 and KMS-5

A high-level and stable expression system of human tissue-type plasminogen activator (t-PA) was accomplished in human cells by selecting a promoter and a host cell line. First, we have constructed two types of t-PA expression plasmids containing 3 kb of the human beta-actin promoter region or 0.3 kb of SV40 early promoter region and these plasmids were transfected into HeLa cells, respectively, and the resulting transfectants were found to secrete various amounts of t-PA derived from the plasmids to the culture media. Southern blot analysis revealed that the beta-actin promoter was more efficient than the SV40 early promoter with regard to the expression level per single copy of the t-PA gene in the transfected HeLa cells. Next, the t-PA expression plasmid containing the beta-actin promoter was also transfected into WI-38 VA13 cells, a human fibroblastic cell line, and KMS-5 cells, a human lymphoid cell line, in order to compare the expression ability of the promoter among these three cell lines. Some of the transfectants from both cell lines were also found to produce t-PA. It was also found that the expression levels in HeLa and WI-38 VA13 seemed to be more efficient than that in KMS-5.     

Dilution enhancement of COS cell expression cloning

To search for an efficient expression cloning method, we mixed plasmid pmDATsv, which contains the mouse dopamine transporter (mDAT) cDNA, with a large amount of another plasmid prGlyTsv to mimic the situation of a cDNA library and examined COS cell expression. Both plasmids have an SV40 replication origin and thus will be replicated to high copy numbers in COS cells. After transfecting COS-7 cells with pmDATsv/prGlyTsv mixture at 1/1000 ratio, we could not detect any cells expressing strong mDAT activity. In contrast, when prGlyTsv was replaced by prSERTsk (no SV40 origin) in the transfection mixture, we observed hundreds of cells expressing strong mDAT activity. The results suggested that in many cells low mDAT expression was not due to the lack of pmDATsv plasmid but due to the presence of large numbers of replicable prGlyTsv. Analysis with a mathematical model suggests that diluting cDNA libraries with other plasmids without the SV40 origin should improve the detection of COS cells expressing target cDNAs. We tested this conclusion with pmDATsv/prGlyTsv mixture. When the mixture at 1/1000 ratio was diluted with prSERTsk and used for transfection, we could now easily detect cells expressing strong mDAT activity.     

High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.     

Transient expression of hepatitis B virus surface antigen (HBsAg) gene in monkey cells by a SV40-based virus vector

Summary We have constructed a recombinant SV40-based vector carrying the S gene coding for the hepatitis B virus surface antigen (HBsAg). This vector replicates as an episome in monkey COS7 cells, producing high levels of hepatitis B virus surface antigen (HBsAg), which is liberated in the cell medium, probably as a membrane vesicule. The vector also carries the SV40-late genes and produces recombinant viruses. These viruses were used to infect fresh cell culture, with detection of HBsAg in the medium. Thus, this virus vector can efficiently transduce the gene for HBsAg.     

High level expression of human tissue-type plasminogen activator gene in mouse C127 cell.

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.     

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.