亚洲小车蝗痘病毒增效因子的初步研究

OaEPV经2×SDS包涵体碱性裂解缓冲液裂解,离心,分别用上清液及沉淀与绿僵菌孢子混合感染黄胫小车蝗,上清液的增效活性比沉淀大2.5倍.用三种不同方法(2×SDS包涵体碱性裂解液法,0.02 mol/L NaOH裂解法,8 mol/L尿素裂解法)裂解OaEPV所得蛋白液与绿僵菌混用,生测结果表明,包涵体裂解液法制备所得蛋白增效活性最高,NaOH裂解法次之,尿素裂解法制备的蛋白几乎没有增效活性.将OaEPV用包涵体碱性裂解液裂解后,用葡聚糖G-200 凝胶柱层析进行分离,出现两个洗脱峰,这两个峰的洗脱液浓缩后进行生物测定,初步表明增效作用主要为第二个峰的蛋白片段.经SDS-聚丙烯酰胺凝胶电泳,第二个峰的蛋白片段的分子量为40 kDa左右.     

颗粒裂解肽G13结构域在大肠杆菌中的高效融合表达

为高效表达颗粒裂解肽G13结构域并避免G13对宿主菌的毒性, 将人工合成的编码G13的基因片段, PCR扩增后克隆于原核表达载体pThioHisA中, 构建了重组表达载体pThioHisA-G13, 将其转化于大肠杆菌BL21(DE3)中, 经IPTG诱导表达融合蛋白Trx-G13, 表达产物以包涵体的形式存在, 其表达量约占细菌总蛋白的58%。包涵体蛋白经 8 mol/L尿素溶解后, 再经CNBr切割, 阳离子交换层析, 得到纯化的重组G13结构域。琼脂糖扩散法检测表明重组G13结构域多肽具有抗菌活性。     

人呼吸道合胞病毒截短F1重组蛋白包涵体的制备、纯化和复性

目的将前期在大肠埃希杆菌中获得表达的A型人呼吸道合胞病毒兰州分离株截短的F1重组蛋白进行纯化和复性,为后期动物免疫制备抗原。方法 37℃诱导重组菌体p ET-42b-F1J/Rossata,诱导完毕后离心收集菌体,高压破碎菌体并收集包涵体后用不同浓度的Triton X-100(细胞裂解液)洗涤包涵体3次。洗涤的包涵体用8 mol/L尿素进行溶解并用镍离子亲和层析方法进行初步纯化,用阳离子交换层析方法对初步纯化蛋白进行最终的纯化。亲和层析纯化蛋白用3种不同的复性液进行了稀释复性。结果 37℃诱导5 000 m L重组菌p ET-42b-F1J/Rossata共收获37 g湿菌体,经过不同浓度Triton X-100洗涤包涵体后纯度可达75%。包涵体用8 mol/L尿素溶解后经镍离子亲和层析纯化纯度约为40%,再用阳离子交换层析介质SP HP进一步纯化样品后纯度可达90%。纯化蛋白以3种不同的复性液都能得到复性,其中复性液3的复性效果相对较好。结论实验中探索了人呼吸道合胞病毒截短F1重组蛋白包涵体的纯化方法及步骤,为后期的蛋白制备及动物免疫奠定了基础。     

重组融合蛋白Trx-IFN-CSP复性工艺研究

旨在建立并优化融合蛋白Trx-IFN-CSP的复性工艺。对重组融合Trx-IFN-CSP进行体外复性研究,考查p H、温度、蛋白浓度、氧化还原体系及辅助复性小分子等复性条件对融合蛋白重折叠的影响。结果显示,适合Trx-IFN-CSP复性的方法为,反复冻融联合超声破菌获得包涵体;用含有1%Triton X-100、2 mol/L尿素、2%DOC洗涤液初步纯化包涵体;再用6 mol/L盐酸胍溶解液变性包涵体;脉冲加样稀释变性液后4℃条件下梯度透析复性,使用L-Arg辅助复性。经肠激酶切去Trx标签后,每升发酵液最终获得110-130 mg肝靶向干扰素,每批蛋白纯度都在95%以上,比活性在1.9-2.4×108 U/mg之间,制备工艺稳定。     

MMP-9-PEX原核表达、纯化及其对结肠癌细胞侵袭能力的影响

用RT-PCR法扩增出基质金属蛋白酶-9(MMP-9)C末端血红素结合蛋白样结构域(PEX),克隆至表达载体pET32a中并转化至E.coilBL21(DE3),经IPTG诱导后在约为42kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量占菌体总蛋白的50%左右。重组蛋白质pET32a/PEX主要以包涵体形式表达,其在上清液中的含量极微。含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包涵体采用金属整合层析有效分离出目标蛋白,纯化后pET32a/PEX蛋白质的纯度大于90%。在Transwell实验中发现复性纯化后的重组蛋白质pET32a/PEX能抑制结肠癌细胞的侵袭,且呈剂量依赖性。     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。     

奶牛γ干扰素基因的高效表达及活性测定

经刀豆素(conA)刺激诱导奶牛外周血淋巴细胞,应用RT-PCR方法从其总RNA中对奶牛γ干扰素基因cDNA进行扩增,然后将特异性片段连接到pMD18-T载体,测序结果表明,与已知序列同源性为100%.然后将特异性片段连在pRLC载体上进行表达,经SDS-PAGE分析,原核表达产物为16kDa的重组蛋白,占菌体总蛋白的42%,表达产物以包涵体形式存在.经7mol/L盐酸胍的变性液溶解及0.5mol/L盐酸胍复性液处理,表达产物进行脱盐、凝胶层析纯化,细胞病变抑制法结果表明,重组牛IFN-γ具有较高的干扰素活性,约为6.0×105U/mg.     

大肠杆菌表达的人重组IL-3的纯化

本文继大肠杆菌表达含凝血酶切点的人重组IL-3融合蛋白成功的基础上进一步探讨了天然型rhIL-3的纯化。超声破碎细菌细胞得包涵体,经洗涤处理可使包涵体纯度达90％,用8mol/L尿素变性溶解包涵体沉淀后直接稀释复性,再超滤浓缩、凝血酶消化,释放天然型rhIL-3。经DEAE Sepharose Fast Flow和Sepharcyl-100 HR层析得到天然型IL-3,纯度达96％,回收率20％以上,具有刺激正常人骨髓细胞形成集落的活性。本实验为大批量重组IL-3的生产创造了条件。     

rhHGFα重组质粒的构建及其在大肠杆菌中的表达

目的 :建立人肝细胞生长因子α链 (rhHGFα)高效原核表达体系 ,分离纯化rhHGFα蛋白 ,检测其生物活性。方法 :以pRC CMV hHGF为模板 ,扩增hHGFαcDNA继以XhoⅠ切为两个片段 ,亚克隆入pBSKS,测序后 ,将上述两个片段与pBV2 2 0连接 ,构建重组质粒。转化大肠杆菌 ,筛选高表达菌株。分离包涵体 ,8mol L尿素溶解 ,稀释复性后纯化重组蛋白 ,MTT法检测活性。结果 :测序证实所克隆的hHGFα基因序列正确 ,筛选出高效表达菌株E .coliBY ,SDS PAGE显示在相对分子量 5 2 0 0 0处出现特异的目的蛋白表达带 ,表达量约占全菌蛋白的 2 5 %。表达产物以不溶性的包涵体 (IB)形式存在 ,复性后具有刺激原代培养成年大鼠肝细胞生长的作用。结论 :以大肠杆菌为宿主 ,成功表达了hHGFα蛋白 ,为进行hHGFα结构与功能研究及中试生产奠定了基础。     

重组中国汉族人源γ-干扰素的纯化和鉴定

在E.coli中高表达的中国汉族人源γ-干扰素是以包涵体的形式存在的,包涵体经各种溶液洗涤后,用8 mol/L尿素裂解,裂解上清在8 mol/L尿素存在下,经离子交换柱层析和凝胶过滤两步纯化,得到纯度大于95%的γ-干扰素,稀释复性后,其比活为5.5×10⁵ U/mg. 激光解析电离质谱分析表明,中国汉族人源γ-干扰素的分子质量为17.32 ku,其N端序列与Gray等报道的γ-干扰素N端序列一致.中国汉族人源γ-干扰素的氨基酸组成分析结果也与其理论值相吻合.     

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.