家蚕抗菌肽CD高效表达及其抗BmNPV感染作用

通过大肠杆菌JM109诱导家蚕,提取其脂肪体总mRNA后,通过RT-PCR得到cDNA,根据GenBank上家蚕抗菌肽CecropinD的cDNA序列,设计并合成引物,然后PCR扩增得到CecropinD肽基因并克隆到pGEM-T载体中,经过EcoRΙ和XhoI酶切,连接并将CecropinD肽基因插入pET32a表达载体中。用重组质粒pET32a-ecropinD转化大肠杆菌BL21(DE3),在IPTG诱导下,融合蛋白Trx-CecropinD以可溶形式得到高效表达,经SDS-PAGE检测显示分子量为23kDa与预期大小相符,表达量约为总蛋白的30%。融合蛋白经Ni2 柱纯化后通过肠激酶切割后释放为Trx(18kDa)和CecropinD(5kDa),最后通过超滤管分离得到重组抗菌肽。通过抑菌实验测得重组CecropinD对于革兰氏阴性及阳性菌均有抑菌活性。并将重组CecropinD与家蚕病毒BmNPV作用混合4h后,一起投喂家蚕,发现病毒感染力有明显降低,说明其有抗病毒感染作用。     

抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

目的是构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的融合蛋白表达载体。从pUC118～hLyso上卸下人溶菌酶基因后,通过重叠区扩增法人工合成抗菌肽B基因,并将其融合到人溶菌酶基因的5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的3’端。PCR鉴定及序列分析表明,所转化的BL21(DE3)菌落中含有插入Cecropin B-hLyso基因的重组质粒pET32a-CB-hLyso。     

利用融合蛋白EDDIE在大肠杆菌中高效表达抗菌肽Cecropin AD

本研究采用猪瘟病毒(Classical swine fever virus,CSFV)定点突变外壳蛋白(EDDIE)为融合蛋白,对抗菌肽Cecropin AD(CAD)基因进行了高效融合表达,获得了有抗菌活性的抗菌肽CAD。首先采用重叠PCR基因合成技术将编码抗菌肽的CAD基因与猪瘟病毒定点突变外壳蛋白EDDIE编码基因合成为e-cad融合基因,接着将融合基因e-cad采用定点同源重组的方法连接到载体pET30a上,构建成pETED表达载体,然后转化大肠杆菌BL21(DE3)表达,表达的融合蛋白在大肠杆菌中主要以包涵体形式存在,表达量占菌体总蛋白的40%以上。蛋白质在体外复性,融合蛋白中EDDIE自我剪切,产生抗菌肽CAD。抑菌试验表明抗菌肽CAD能有效地抑制大肠杆菌和藤黄八叠球菌的生长,并且对酵母菌的生长也有微弱地抑制作用。以EDDIE为融合蛋白是在大肠杆菌中高效表达抗菌肽的一种好方法。     

抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

家蝇防御素在大肠杆菌中的表达、纯化与抗体制备

家蝇防御素是从家蝇中克隆得到的1种抗菌肽。为了进一步研究家蝇防御素的功能和制备特异性抗体,采用大肠杆菌表达外源蛋白的方法, 进行了家蝇防御素原核表达的研究。根据克隆到的家蝇防御素基因(Mdde) 的cDNA序列, 设计特异性引物, PCR 扩增成熟肽的cDNA片段, 将成熟肽序列重组到表达载体pGEX 4T 1中, 构建m Mdde/pGEX 4T 1重组表达载体, 在大肠杆菌BL21 中诱导表达, 重组表达的融合蛋白GST Mdde占菌体总蛋白的33 4%。纯化得到GST Mdde后, 再用凝血酶将其从特定位点切开, 得到表达的m Mdde。液体抑菌实验结果初步表明, 表达的融合蛋白GST Mdde对细菌生长有一定的抑制作用。利用纯化的GST Mdde融合蛋白, 制备了抗血清。     

A型口蹄疫病毒VP0基因的克隆及原核表达

从实验室冻存的含A型口蹄疫病毒的细胞中提取FMDV总RNA,通过RT-PCR获得cDNA.并根据FMDV全基因组序列设计了一对针对VP0基因的引物,通过PCR扩增得到目的基因VP0并亚克隆入pMD18-T载体.将鉴定出的阳性质粒和表达载体pET32a用BamH Ⅰ和HindⅢ双酶切回收后连接获得阳性重组质粒pET32-VP0.用IPTG诱导重组质粒表达目的蛋白VP0并用SDS-PAGE进行检测.表达产物用镍亲和树脂进行了纯化.结果证明,口蹄疫病毒VP0蛋白在大肠杆菌中获得了高效表达且表达产物得到了纯化,为实验室进一步的研究提供了重要的材料.     

人IL-3基因原核表达载体的构建及表达

目的：构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法：通过佛波酯（TPA）和植物球血凝素（PHA）刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应（RT-PCR）获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a（＋）中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷（IPTG）诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果：酶切鉴定和测序结果证明成功构建了原核表达载体pET32a．IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论：成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。     

人IL-3基因原核表达载体的构建及表达

目的:构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法:通过佛波酯(TPA)和植物球血凝素(PHA)刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应(RT-PCR)获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a(+)中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果:酶切鉴定和测序结果证明成功构建了原核表达载体pET32a-IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论:成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。     

鲎血细胞中脂多糖结合蛋白TALF在大肠杆菌中的表达研究

TALF(Tachyleus antilipoposaccharide factor)对细菌内毒素（LPS）的核心部分有抑制作用。研究TALF cDNA基因在大肠杆菌中的表达,首先将TALF cDNA基因分别插入大肠杆菌表达载体pGEX-4T-2、pET22b、pET28a中,构建重组表达质粒,转化于大肠杆菌BL21(DE3)。结果表明克隆于pET22b、pET28a中的TALF cDNA基因没有表达,而融合了GST的TALF基因（GST-TALF）能够在大肠杆菌中表达,并形成包涵体。从1L培养基中可获得4mg纯度为91%的GST-TALF融合蛋白。经复性和纯化后的融合蛋白GST-TALF几乎检测不到抑菌活性及LPS中和活性,但该融合蛋白经凝血酶消化后表现出明显的体外抑菌活性及LPS中和活性。     

新型抗菌肽Perinerin在大肠杆菌中的融合表达

为了获得新型抗菌肽 perinerin在大肠杆菌中的高效和可溶表达,实验首先采用SOE 法(重叠 PCR 法)获得的 perinerin 基因序列,对目的基因密码子优化,然后将其连接到 pET32a 载体中获得重组表达载体 pET32a-PEN,通过改变诱导时间和温度、诱导剂 IPTG 浓度以及诱变工程菌株等条件和方法,观察重组蛋白的表达效果,并运用金属螯合层析对融合蛋白进行纯化.SDS-PAGE 显示重组菌诱导后表达的融合蛋白分子量约为 26kD,采用变异重组菌株 MUT 3诱导表达,在 2×YT 培养基培养条件下,30 ℃诱导4 h 可获得高效表达的 perinerin 融合蛋白,其表达量约占菌体总蛋白的 50 % 左右,重组蛋白主要以可溶性表达形式存在,可溶性产物最高可达重组蛋白总表达量的 60 %.融合蛋白运用金属螯合层析一步纯化,纯度可达 90 % 以上.     

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.