Ribozyme对癌基因Ki-ras~(G12V) mRNA的剪切及其特异性

目的 :设计切割ki-rasG12V mRNA的特异性ribozyme(Rz2 17) ,明确其对癌基因ki-rasG12VmRNA的细胞内外切割活性 ,为以ki-rasG12VmRNA为特异性靶分子的基因治疗及癌基因ki-ras的功能研究提拱一种新的途径。方法 :依Symons总结的“锤头结构”原理 ,设计一种能特异性切割ki-rasG12VmRNA的ribozyme ,利用DNA重组技术构建ki-rasG12V外显子 1和ri-bozymeRz2l7的体外转录质粒及ribozymeRz2 17的真核表达质粒 ,体外转录获得ribozymeRz2 17及ki-rasG12V外显子 1mRNA ,在含Mg2 溶液中ribozymeRz2 17对其靶RNA分子进行切割。以RT -PCR对转染ribozymeRz2 17真核表达质粒的细胞ki-rasG12VmRNA进行半定量分析。结果 :ki-rasG12V外显子 1体外转录mRNA分子 ,能被ribozymeRz2 17定点切割而野生型ki-ras外显子 1体外转录mRNA则不被切割 ;转染ribozymeRz2 17的胰癌细胞ki-rasG12VmRNA含量减少 ,而转染ri bozymeRz2 17的肝癌细胞其内源性ki-rasmRNA含量无明显变化。结论 :ribozymeRz2 17无论在细胞内外均能剪切突变型ki-rasmRNA(G12V)而且其切割作用为突变型ki-rasG12VmRNA特异性的。     

A study on improvement of expression of human G－CSF cDNA transferred with retroviral double－copy vector

ＡｓｔｕｄｙｏｎｉｍｐｒｏｖｅｍｅｎｔｏｆｅｘｐｒｅｓｓｉｏｎｏｆｈｕｍａｎＧ－ＣＳＦｃＤＮＡｔｒａｎｓｆｅｒｒｅｄｗｉｔｈｒｅｔｒｏｖｉｒａｌｄｏｕｂｌｅ－ｃｏｐｙｖｅｃｔｏｒＧｕｏＢａｏｙｕ（郭葆玉）；ＺｈａｎｇＳｕｙｉｎｇ（张淑英）；ＸｕＨｕｉ...     

Evaluation of a sulfonated DNA probe (sulfoprobe) for diagnosis of falciparum malaria

In this study,the DNA probe pPF14 was nonradioactively labelled by sulfo-modifica-tion,and used in a dot blot hybridization assay for detection of P.falciparum.The resultsshowed that the sulfoprobe successfully detected 25pg purified DNA and 0.001% parasitemia ofcultured P.falciparum,and did not react with human leukocyte DNA.In the study of 179clinical blood samples of malaria patients from Yunnan province,the DNA probe results corre-lated well with blood smear ones.Of 107 P.falciparum samples determined by microscope ex-amination,99 were positive by sulfoprobe (92.5%);Of 72 P.vivax samples,1 was crosslyreacted;no cross reactions were found with any of 48 normal blood samples.It is indicated thatsulfoprobe may be useful in specific diagnosis and epidemiological investigation of P.falciparuminfection.     

疟原虫裂殖子抗原分析

疟疾是严重危害人类健康的疾病,疟疾疫苗是很有效的防治手段。研制疟疾疫苗,首先要对疟原虫抗原进行分析、鉴定。疟原虫裂殖子直接暴露于机体免疫系统,对裂殖子保护性抗原的分析、鉴定是疟疾疫苗研制的关键部分。随着免疫学和分子生物学技术的发展,已对许多疟原虫裂殖子保护性抗原进行了分子水平的研究,取得了可喜的进展,特别是对gp195、83KDa、75KDa等主要裂殖子表面抗原进行了核苷酸序列分析和免疫保护作用的研究,为疟疾疫苗的研制提供了基础。     

基因重组肿瘤坏死因子对LAK细胞体内外抗肿瘤作用的增强效果

TNF单独不能诱导LAK活性,也不能进一步提高最适剂量IL-2(1000U/ml)诱导的LAK活性,但能显著增强亚适剂量IL-2(10～100U/ml)诱导的LAK活性。抗TNF单抗和抗IL-2受体β链(p75)单抗显著抑制TNF/IL-2对LAK活性的协同诱导作用。实验证明,TNF能显著增强IL-2/LAK细胞的体内抗肿瘤效果,对于腹水型肝癌小鼠,以联合腹腔内注射TNF/IL-2/LAK细胞的体内抗肿瘤效果最佳。     

腺相关病毒末端反向重复顺序折叠结构在真核细胞染色体基因组上的整合作用

腺相关病毒（Ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄｖｉｒｕｓ，ＡＡＶ）是一类线状单股ＤＮＡ（ＳＳ－ＤＮＡ）的微小病毒。依据Ｃａｖａｌｉｅｒ－Ｓｍｉｔｈ的ＳＳ－ＤＮＡ病毒复制模型原理，将其末端反向重复顺序（ＩＴＲｓ）分离，变为折叠十字架结构，再以其发夹中的３＇－ＯＨ为引物合成另一股姊妹染色体ＤＮＡ，然后共价连接到亲代分子上，与辅助病毒Ａｄ２和ＰＡＡＶ／Ａｄ共转染真核生物细胞。Ｓｏｕｔｈｅｒｎ印迹分子杂交结果表明：插入这一折叠结构中由ＳＶ４０早期启动子控制的报告基因－新霉素抗性基因拷贝已整合到细胞基因组染色体上，这种以无细菌ＤＮＡ分子为支架的折叠结构不仅证明了Ｃａｖａｌｉｅｒ－Ｓｍｉｔｈ的理论，而且可望为外源基因导入真核细胞提供新的又一系统。     

结直肠癌P16基因点突变的研究

目的 探讨结、直肠癌P16基因突变与癌发生、演进的关系。方法 选结、直肠癌组织、癌旁正常组织42例和7例转移淋巴结,用蛋白酶K消化,提取DNA,用多聚酶链式反应-单链构象多态分析(PCR-SSCP)及DNA测序方法分析P16基因第二外显子的突变。结果 42例癌组织中发现7例有异常泳动条带,占16.7％。在7例癌转移的淋巴结中发现3例异常泳动条带。癌旁组织中未发现异常泳动条带。第二外显子42～61,147～168编码区的核酸出现改变,为碱基G-T、T-G、G-A的碱基转换。突变的发生率与组织分型无明显相关。Duke'C期突变率高于A、B期,有淋巴结转移者突变率也高。结论 P16基因的丧失,可引起Cdk 4活性的负调节,导致细胞无限增殖。本组资料发现点突变在癌组织中占16.7％,转移淋巴结中也有。由于采用了自身正常组织对照,避免了正常多态性的干扰,结果较为可靠。P16基因的改变与临床分期及预后有一定关系。     

Subcloning,sequencing and expressing of conserved blocks of P190 gene of Plasmodium falciparum FCC1/HN strain

190-kilodalton glycoprotein (P190) of Plasmodium falciparum,precursor of themajor surface protein of merozoites,is considered a promising candidate for blood stage malarialvaccine.Six primers were designed according to the sequence of MAD20 strain,with a GCclamp and BamH Ⅰ site at the 5'-end of each one,and a GC clamp and Xba Ⅰ site at the 3'-endof each one.The primers were synthesized by phosphoramidite approach (User's Manual ofABI Company) and purified using HPLC.Three fragments in the second,third and fourth con-served regions of P190 gene of Plasrnodium falciparum FCC1/HN strain isolated from theblood of patients in Hainan Province of China were amplified by the polymerase chain reaction(PCR).The amplified fragments were suhcloned into pUC18 vectors and sequenced using thedideoxy chain termination method.All three regions of P190 gene of FCC1/HN strain also werehighly conservative as compared with P190 gene of MAD20 (Papua New Guinea isolate),K1(Thailand isolate),Wellcome (West Africa isolate) and CAMP (Malaysia) strains ofPlasmodium falciparum.The C at position 81 in the second conserved block of P190 gene ofFCC1/HN isolate was substituted by T,which did not change the amino acid determined by thecodon corresponding to the substitution.The genes sequenced were cloned into rpGEX-2T,aglutathione S-transferase gene fusion system for expression.     

重组人α8干扰素在大肠杆菌中的克隆与高表达

利用基因重组技术构建了人干扰素α８（ＩＦＮ－α８）高表达菌株Ｅ．ｃｏｌｉＸＬｌ－Ｂｌｕｅ／ｐＢｍ，经酶切鉴定、ＤＮＡ测序、ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ及生物活性测定等分析，表明能特异性地表达具有生物活性的ＩＦＮ－α８，表达量达到总菌体蛋白的２３％，经复性后比活为４．８×１０７ＩＵ／ｍｇ，具有良好的应用前景。