重组人核苷二磷酸激酶A的理化性质

对重组人核苷二磷酸激酶A（rhNDPK-A）进行纯化,并对重组产物的理化性质及在溶液中的聚合状态进行鉴定。NDPK-A工程菌发酵后的菌体高压匀浆,然后微孔过滤、超滤浓缩,所得样品经DEAE阴离子交换、Cibacron Blue亲和层析、分子筛层析三步纯化后,以SDS-PAGE和RP-HPLC分析纯化产物的纯度,RP-HPLC测定酶活性。合格制品以基质辅助激光解析飞行时间质谱测定相对分子质量（MW）;Edman降解法测定N末端序列;多角度激光散射法测定重组产物在溶液中的表观分子量。结果表明,rhNDPK-A纯化产物的SDS-PAGE纯度为97.3%,RP-HPLC纯度为99.2%;比活性为（900±100）u/mg;单体相对分子质量为17017,与NDPKA分子量理论值相差132。测序结果表明,rhNDPK-A N末端缺失Met残基,其理论分子量为17017,与飞行质谱测定结果完全一致。表观分子量测定结果表明,rhNDPK-A在溶液中形成六聚体,表观分子量为102kD。上述结果说明, NDPK-A重组产物具与天然产物相同的自发形成六聚体性质,这为NDPK-A新药开发和机理研究打下了良好基础。     

50L规模中试发酵重组核苷二磷酸激酶A工程菌

中试规模发酵重组人核苷二磷酸激酶A（rhNDPK-A）工程菌,并对表达产物进行纯化。摇瓶培养一级种子至合适密度,以10%比例接种二级种子培养基,在7L发酵罐中培养至OD600为9.6～10.5,然后转入80L发酵罐中进行补料分批培养,所得菌体裂解后,经离子交换层析和亲和层析两步纯化得重组蛋白制品。结果表明,50L培养液经过10h培养后,湿菌收量为1560 g/批,NDPK-A表达量为23.8%。另外,补料方式对发酵密度有明显影响。与单纯补加碳源相比,同时补加碳源和氮源可以显著提高菌体产量,但对目的蛋白表达量地提高不明显。在较优条件下,菌体产量为(2220.00±169.71) g/批,蛋白表达量为(22.00±0.42) %,纯化后重组蛋白得率为510mg/L。产物可溶、密度适中、工艺简便的中试发酵条件的建立为高得率、大规模制备重组rhNDPK-A奠定了基础。     

重组点状产气单胞菌脯氨酰内肽酶的纯化和鉴定

报道重组点状产气单胞菌脯氨酰内肽酶(简称apPEP)的基因工程下游工艺研究。工程菌株E.coli BL21/pKKH\|PEP表达产物apPEP为可溶性蛋白,在NBS BioFlo 3000型5L自控发酵罐中经14h培养每升发酵液可达到22.5g干重菌体,含apPEP 3.0g左右。发酵菌体经超声破碎、硫酸铵沉淀后,依次经Sephadex G-25、High performance Q sepharose FF(HP\|Q)、Phenyl separose 6 FF柱层析分离纯化,每升发酵产物最终可得0.86g纯度达96%的重组apPEP,比活力达到65.5u/mg,整个纯化工艺的蛋白回收率为8.2%,活力回收率为24.4%。纯化的apPEP经电喷雾质谱测定分子量为76464±30D,N端氨基酸序列与基因序列推导的一致。等电点为pI6.0左右。与Aeromonas hydrophila来源的PEP(pI=5.5)相近。     

人β-干扰素粗制品的浓缩与纯化

本文报道了采用Millipore超滤膜浓缩人β-干扰素粗制品,滤速快,活性回收率高,同时又具有一定的纯化作用。浓缩后的干扰素样品,再经过一次蓝色葡聚糖——Sepharose CL-6B柱层析,可将其纯化2800倍,特异活性可达10⁶μ／mg蛋白以上的国际临床标准。     

旋毛虫基因重组抗原的纯化

根据旋毛虫基因重组抗原蛋白的特点,筛选出裂解处理工程菌菌体的方法,并探索了用ＳｅｐｈａｃｒｙｌＳ－３００（ＨＲ）柱层析纯化重组蛋白的程序。用本法处理和纯化后,重组蛋白的纯度可达９０％以上。所纯化的三种重组抗原蛋白均能与猪旋毛虫病血清发生特异性反应而不与正常猪血清和猪囊虫病血清反应,其中以重组蛋白ＲＰ３４的特异性最强,ＲＰ３７较弱,ＲＰ４６介于两者之间。研究结果表明,本纯化方法易于操作、设备简单和特异性蛋白回收率高,是处理和纯化旋毛虫基因重组抗原的最佳程序。     

高纯度白介素-3与假单胞菌外毒素融合蛋白的表达、纯化及质谱鉴定

旨在利用简单的表达纯化工艺,获得纯度较高、有活性的重组免疫毒素IL3-PE38KDEL并对其物理学及免疫学性质进行鉴定。利用PQE30-IL3-PE38KDEL/SG12009工程菌表达重组免疫毒素IL3-PE38KDEL,在提取包涵体后经一步强阳离子柱层析得到纯度较高的免疫毒素复性纯化产物,用Westenblotting、质谱、氨基测序等先进技术对产物鉴定。结果显示,发酵后目的蛋白表达量占总菌体的17.56%,经强阳离子柱层析纯化复性后的目的蛋白纯度达到90%以上,Western blotting、质谱、氨基测序结果均表明,纯化产物分子量为54.9kD且具有相应的免疫学活性,序列与预期序列完全一致。纯化复性方法对重组免疫毒素IL3-PE38KDEL的生物性质及纯度有较大影响,因此选择一种适当的纯化复性方法至关重要。     

链球菌溶血素“O”纯化的研究

乙型链球菌３２１７２ 株接种于含有月示蛋白胨的牛肉水培养基中培养。得到含有溶血素“Ｏ”的培养液，其溶血效价２５６ ＩＵ／ｍ ｌ。比活为１１．６ ＩＵ／ｍ ｇ 蛋白。经过超滤除去９５％ 的非目的蛋白，所得超滤截留液的溶血效价为２０４８ ＩＵ／ｍ ｌ，比活为３３５．７ ＩＵ／ｍ ｇ 蛋白。在此基础上用离子交换柱层析纯化，所得活性峰溶血效价为８１９２ ＩＵ／ｍ ｌ，比活为４３１１．５ ＩＵ／ｍ ｇ 蛋白。通过以上两步的分级纯化，溶血素“Ｏ”的比活提高了３７１ 倍。此纯化蛋白在ＳＤＳ－ＰＡＧＥ电泳中呈现一条染色带。     

大肠杆菌发酵生产重组人颗粒溶素

目的:在FUS-50L生物反应器中利用补料分批培养技术培养表达含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS株,生产重组颗粒溶素(GNLY)。方法:选取含pET28a( )-GNLY的BL21(DE3)pLysS菌株单个克隆分级培养,将制备的二级种子液接种于发酵罐中。在发酵过程中,控制溶氧为30%～50%,温度为37℃;在基础培养基内生长4h后,补加以甘油为碳源的补料,继续生长到11h;加入葡萄糖至终浓度为1%,30℃诱导表达6h;收集菌体,纯化制备目的蛋白。利用Western印迹检测重组蛋白的抗原性,用CFU方法检测其生物学活性。结果:发酵液中最终菌体密度达80g/L;纯化所得重组蛋白约占菌体总蛋白的5%,含量为60mg/L;经鉴定所获重组颗粒溶素有较好的免疫学活性和生物学活性。结论:用含重组质粒pET28a( )-GNLY的大肠杆菌BL21(DE3)pLysS表达系统,可得到具有生物活性的重组颗粒溶素,为大批量生产提供了条件。     

甲醇酵母分泌表达重组人干扰素α1b纯化工艺的建立

建立适合大规模、低成本生产重组人干扰素α1b(Recombinanthumaninterferon α1b ,rhIFN α1b)的纯化工艺。采用高效分泌表达rhIFN α1b的甲醇酵母工程菌发酵 ,收集离心后的上清液 ,超滤脱盐 ,经离子交换柱和分子筛柱层析纯化。纯化的rhIFN α1b的纯度为 98%以上 ,比活性 2 .4× 10 7IU/mg ,活性回收率 14 % ,相对分子质量 1980 0和等电点 5 .0。经检测 ,rhIFN α1b蛋白N 端 15个氨基酸序列与正常对照完全符合。该纯化工艺简便 ,时程短 ,重复性好 ,适合于大规模生产     

层析法去除重组人血清白蛋白干扰素a2b融合蛋白的内毒素

用柱层析方法在生产重组人血清白蛋白干扰素α2b融合蛋白过程中去除产品中的内毒素。所用柱层析组合为Blue-sepharose亲和柱层析、SOURCE 15 ISO疏水柱层析、Q Sepharose F.F.离子交换柱层析、Sephadex G25 Coarse凝胶过滤柱。采用鲎试剂法检测柱层析各阶段得到蛋白中的内毒素含量; 并用RP-HPLC方法测定柱层析各阶段得到蛋白的纯度与浓度, 求出每毫克蛋白的内毒素含量。结果为每步柱层析过程式均有去除内毒素的作用, 最终所得蛋白的内毒素含量降为1 EU/mg, 去除率达到99.9%。因而生产重组人血清白蛋白干扰素a2b融合蛋白所用分离纯化柱层析技术在纯化蛋白的同时能有效的去除产品中内毒素, 所得产品内毒素的含量远低于药典对注射剂的内毒素含量要求。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.