人组蛋白H3真核表达载体的构建及功能初步检测

目的构建带myc标签的人组蛋白H3基因真核表达载体,获得Myc-H3融合表达蛋白,并对其功能进行初步检测。方法采用PCR技术从乳腺文库中扩增人H3基因编码序列,将其正确插入pXJ-40-myc载体,重组质粒转染人胚肾293T细胞,Western blot法检测融合蛋白表达,通过免疫共沉淀技术检测融合蛋白与已知结合蛋白组蛋白赖氨酸特异性去甲基化酶(LSD1)和组蛋白甲基转移酶(EZH2)的相互作用。结果构建得到组蛋白H3基因的真核表达载体,双酶切鉴定得到与预期片段大小相符的外源基因插入片段,经测序与目的序列完全一致;经Western blot法检测,融合蛋白成功表达;免疫共沉淀检测证实Myc-H3融合蛋白可以和已知相互作用蛋白LSD1及EZH2相互作用。结论成功构建组蛋白H3真核表达载体,该融合蛋白正确表达。     

鸡蛋主要过敏原Gal d1片段基因的克隆、表达、纯化及免疫学鉴定

目的克隆、表达和纯化鸡蛋主要过敏原Gald1区基因,并对其免疫学活性进行鉴定。方法用生物信息学方法对鸡蛋主要过敏原Gald1基因进行抗原表位预测,设计特异性引物,用PCR的方法分别扩增Gald1N端和C端基因,将其分别连接到原核表达载体PET32a上。重组质粒转入大肠杆菌,用IPTG诱导表达,通过镍亲和柱层析法纯化重组蛋白,最后用Western-blotting检测重组蛋白的免疫原性。结果表达的蛋白均以可溶性为主,N端融合蛋白相对分子质量为28600,C端融合蛋白相对分子质量为26800,纯化的N端和C端蛋白均有较好的免疫原性,C端的免疫原性强于N端。结论成功地克隆和表达了鸡蛋主要变应原基因Gald1的N端和C端,为鸡蛋过敏的诊断和免疫治疗奠定了基础。     

采用反向遗传学技术构建H5亚型禽流感疫苗株

目的构建重组H5亚型禽流感疫苗株。方法采用RT-PCR技术，分别扩增鹅源高产禽流感病毒A／Goose／Dalian／3／01（H9N2）的6条内部基因片段、高致病性禽流感病毒株A／Goose／HLJ／QFY／04（H5N1）的血凝素（HA）基因和N3亚型参考株A／Duck／Germany／1215／73（H2N3）的神经氨酸酶（NA）基因，并对HA1和HA2连接肽处的5个碱性氨基酸（R-R-R-K-K）的编码序列进行缺失与修饰，然后分别构建这8个基因的转录与表达载体，将其共转染293T／MDCK混合培养细胞单层，对拯救出的重组病毒进行表型分析。结果利用反向遗传学技术拯救出了全部基因都源于禽流感病毒的疫苗株，其基因序列符合设计要求包括删除HA基因的毒力相关序列，疫苗株的表型为H5N3。结论构建成功重组禽流感疫苗株rH5N3，为制备H5亚型禽流感疫苗打下了坚实的基础。     

抗人HBsAg单链抗体基因的构建及其在COS-7细胞中的表达

目的：构建抗人HBsAg单链抗体基因，并分析其在COS—7细胞中的表达。方法：以从噬菌体抗体库中筛选的抗HBsAg的Fab抗体基因为模板，分别扩增其轻、重链可变区(VL、VH)基因，通过重组PCR方法将轻、重链可变区基因用连接肽(C1y4Ser)3的编码序列连接，并引入前导肽编码序列，构建具有L—VH—Linker—Vl结构的单链抗体基因。将所构建的单链抗体基因克隆入绿色荧光蛋白(GFP)融合表达载体pEGFP—N3，并转染COS—7细胞进行表达。结果：经测序表明，前导肽、连接肽、VL及Vh的序列正确。酶切鉴定证实，成功地构建了GFP基因融合表达载体。瞬时转染COS—7细胞后，通过荧光显微镜观察证实有ScFv融合蛋白的表达。转染细胞的培养上清浓缩后，进行SDS—PAGE及westem blot分析，可检出ScFv融合蛋白的分泌性表达。培养上清的间接ELISA检测证实，所表达的单链抗体具有与HBsAg结合的特异性。结论：成功地构建了抗人HBsAg单链抗体基因，并可在COS—7细胞中分泌性表达。     

sea基因定位突变体构建及其重组表达产物生物学活性的研究

目的 构建葡萄球菌肠毒素A（SEA）基因及其突变体原核表达系统，了解突变前后重组表达产物的细胞毒性、促淋巴细胞增殖和抑制肿瘤生长活性的变化。方法采用高保真PCR和TA克隆法，从金黄葡萄球菌ATCC13565株DNA中扩增并克隆了不含信号肽序列的湖基因。采用突变引物PCR构建sea基因定位突变体。建立sea基因及其定位突变体原核表达系统。Ni—NTA亲和层析法提纯表达的目的重组蛋白。采用TCID50。法测定目的重组蛋白对Vero细胞的细胞毒性。采用MTT比色法分别检测不同浓度的目的重组蛋白促小鼠脾细胞增殖及对抑制KB和HL-60癌细胞株生长的作用。结果 所克隆的sea基因核苷酸序列与报道序列的相似性为100％，7种突变体均在既定位置获得预期的密码子突变。rSEA和各突变体重组蛋白表达量约为细菌总蛋白的52％。rSEA对Vero细胞TCID50为4．1μg，其突变体重组蛋白分别为5．8～23．5μg。1和5μg/ml的rSEA及其5种突变体重组蛋白rSEA／H187A、rSEA／H225A、rSEA／D227A、rSEA／H187A／D227A和rSEA／H225A／D227A对小鼠脾细胞均有明显的促增殖作用（P〈0．05），10和20μg/ml的rSEA及上述5种突变体重组蛋白促小鼠脾细胞增殖活性与100μg/ml PHA相似（P〉0．05）。5～20μg/ml的rSEA及上述5种突变体重组蛋白作用的小鼠脾细胞上清及其上清与脾细胞混合物均能有效地抑制KB和HL-60细胞生长（P〈0．05）。结论 本研究成功地构建了sea基因突变体及其高效原核表达系统，其表达产物的细胞毒性均有所降低。由于rSEA／H225A、rSEA／D227A、rSEA／H225A／D227A细胞毒性较低、促脾细胞增殖和抑制肿瘤细胞生长活性较强，可作为研制升白细胞、抗肿瘤SEA相关药物的候选突变体。     

肠出血性大肠埃希菌O157:H7 espA基因的克隆与表达

目的 通过DNA重组技术表达肠出血性大肠杆菌（EHEC）O157：H7的EspA蛋白，并分析其免疫学性质。方法 采用PCR技术从EHEC O157：H7基因组中扩增espA基因，T／A法克隆，连接至pET-28a（＋）载体上，转化宿主细胞E．coli BL21（DE3），IPTG诱导表达，亲和层析法纯化目的蛋白，SDS-PAGE测定其相对分子质量，同时分析其N端氨基酸序列，免疫家兔鉴定其抗原性。结果 重组espA基因片段的测序结果与GenBank上基因序列只有1个碱基不同，一致性为99．83％，所编码的氨基酸序列没有改变。重组EspA蛋白在工程菌中以包涵体的形式表达，表达量约30％。免疫家兔所得抗体滴度为1：32。结论构建高效表达EspA蛋白的重组载体pET-28a（＋）-espA，表达的蛋白具有良好的免疫原性，为进一步制备亚单位疫苗奠定基础。     

核小体Th表位和CTLA4Ig双基因共表达真核表达载体的构建

目的将核小体Th表位与CTLA4Ig融合基因融合，研究CTLA4Ig作为真核表达载体的可行性。方法用touchdown PCR法扩增CTLA4Ig基因，同时引入核小体Th表位（H2B14-28）。将PCR产物连接真核表达载体pcDNA3．1（＋），构建pcDNA3．1（＋）-CTLA4Ig—H2B。将表达质粒转染COS-7细胞，Western blot检测转染细胞裂解上清中融合蛋白的表达。将构建表达CTLA4Ig—H2B的减毒鼠伤寒沙门氏菌SL7207喂饲BALB／c小鼠，取脾脏进行免疫组化鉴定重组蛋白在动物体内的表达。结果酶切鉴定和基因序列测定显示重组质粒构建成功。在pcDNA3．1（＋）-CTLA4Ig—H2B质粒转染后48h细胞裂解上清中，检测到CTLA4Ig融合蛋白的表达，该蛋白能与抗人CTLA-4单抗特异结合。重组蛋白在BALB／c小鼠脾脏免疫细胞胞浆中有阳性表达。结论成功构建了能稳定表达核小体Th表位和CTLA4Ig融合基因的真核表达载体。     

HIV—1gp41基因的分段克隆和表达

目的 在大肠杆菌中表达HIV－1gp41N肽和C肽基因。方法 用PCR方法从含HIV－1gp160基因的质粒中扩增HIV－1gp41N肽和C肽基因,重组人pGEX-4T-1载体,并亚克隆人pGEM7zf( )中测定核苷酸序列,限制性酶切鉴定后进行原核表达。结果 成功地扩增到HIV－1gp41N肽和C肽基因,酶切鉴定及测序结果与已知HIV－1亚型的gp41N肽区和C肽区基因序列一致,SDS－PAGE结果显示,表达出与预期分子量大小相同的蛋白。结论 N肽和C肽基因的成功表达,为进一步研究其结构和功能奠定了基础。     

组蛋白H3K9甲基化的多重作用

组蛋白是染色质的核心，其尾部共价修饰在基因表达调控中有重要作用。赖氨酸甲基化是组蛋白共价修饰之一，在多种生物学过程有重要作用。Suv39h1作为第一个被发现的组蛋白赖氨酸甲基转移酶，可以催化H3K9甲基化。H3K9甲基化是异染色质蛋白（HP1）染色区的停泊位点（docking site)。研究表明，组蛋白H3K9甲基化在异染色质形成及基因转录调控中具有重要的作用。     

禽流感病毒(H5N1)血凝素蛋白真核表达载体的构建与表达

目的:构建禽流感病毒(H5N1)血凝素蛋白HA真核表达载体并表达其编码蛋白(转染293T细胞)。方法:从江苏H5N1流感病毒毒株(A/Jiangsu/07-4(H5N1))提取病毒RNA,采用RT-PCR技术扩增HA全长基因,将其克隆至pMD18-T Vector中构建pMD18-T-HA质粒,双酶切pMD18-T-HA与PXJ40-MYC后,构建真核表达载体PXJ40-MYC-HA,经酶切及测序鉴定后将质粒转染到293T细胞中,通过West-ern blot鉴定HA蛋白的表达。结果:经双酶切、测序鉴定证实HA基因的真核表达载体构建成功。Western blot法可见HA基因编码蛋白的成功表达。结论:成功构建了H5N1血凝素真核表达载体,该表达载体的构建为后期建立稳定表达HA蛋白的细胞模型和HA蛋白功能研究奠定了基础。     

Transferrin and the innate immune response of fish: identification of a novel mechanism of macrophage activation

We have previously demonstrated that a non-cytokine serum protein called transferrin was a primary activating molecule of the goldfish (Carassius auratus) macrophage antimicrobial response. The ability of the enzymatically cleaved forms of this protein to modulate fish macrophage function is novel and may represent a primitive and evolutionary conserved mechanism for the induction of NO response of macrophages. In the present study we confirm our earlier findings using immunoaffinity purified goldfish transferrin from mitogen-stimulated leukocyte supernatants. In addition we demonstrate that: (1). products released by necrotic/damaged cells contain transferrin-cleaving activity; (2). the cleavage site is located within the bridge peptide connecting the two lobes of the transferrin molecule; (3). transferrin is expressed by activated goldfish macrophages but not mitogen-stimulated kidney leukocytes; and (4). addition of transferrin significantly enhanced the killing response of goldfish macrophages exposed to different pathogens or pathogen products (e.g. lipopolysaccharide, Mycobacterium chelonei, Trypanosoma danilewskyi, Aeromonas salmonicida, and Leishmania major). We propose a model of fish macrophage activation that is mediated by a non-cytokine host protein (i.e. transferrin) in combination with highly conserved innate immunity recognition receptors that are almost certain to exist in teleost.     

Adrenomedullin and proadrenomudullin N-terminal 20 peptide (PAMP) are present in human colonic epithelia and exert an antimicrobial effect

The hypotensive and vasorelaxing peptides adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), were found to be distributed on the surface of the colonic mucosa. AM and PAMP showed dose-dependent antimicrobial activity against E. coli. The results suggest that the novel vasoactive peptides AM and PAMP play an important role in mucosal defence.     

Isolation and characterisation of oncorhyncin II,a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout,Oncorhynchus mykiss

A potent antimicrobial peptide, tentatively named oncorhyncin II, was isolated from an acid extract of rainbow trout skin secretions. Amino acid sequencing showed that the first 17 residues of oncorhyncin II are identical to residues 138-154 of histone H1 from rainbow trout. Matrix-assisted laser desorption ionization mass spectrometry revealed that the purified peptide has a molecular mass of 7195.3Da. Taken together, these data indicate that oncorhyncin II is a 69-residue C-terminal fragment of histone H1, probably phosphorylated at two residues. Oncorhyncin II has minimal inhibitory concentrations in the submicromolar range against Gram-(+) as well as Gram-(-) bacteria and it does not display significant haemolytic activity towards trout erythrocytes. The purified peptide was found to induce a marked destabilisation of planar lipid bilayers without the formation of stable ion channels. Oncorhyncin II is possibly a cleavage product of histone H1 with a potentially important role in mucosal defence of rainbow trout.     

Isolation of human cationic antimicrobial protein-18 from seminal plasma and its association with prostasomes

BACKGROUND: Cathelicidins are a group of antibiotic peptides with broad antimicrobial activity. They are considered to be an essential part of the innate immune system. The only known human cathelicidin is the human cationic antimicrobial protein (hCAP-18), from which the antimicrobial peptide LL-37 is released. METHODS AND RESULTS: In the present study, we purified hCAP-18 from seminal plasma and confirmed its identity by N-terminal amino acid sequencing. Gel filtration of seminal plasma showed the presence of hCAP-18 in both a low and a high molecular weight peak. Fractions corresponding to the high molecular form of hCAP-18 also contained dipeptidyl peptidase IV (CD26), a prostasome marker. This finding suggested that hCAP-18 found in fractions corresponding to high molecular weight molecules, is prostasome-associated. Flow cytometry confirmed the association of hCAP-18 with prostasomes and indicated that the molecule is surface bound. Western blot showed the presence of intact hCAP-18 in sperm, prostasomes and ultracentrifuged seminal plasma. CONCLUSIONS: These findings suggest that hCAP-18 may have an important role in antimicrobial defence during human reproduction. The binding of hCAP-18 to prostasomes indicates that protasomes can serve as a reservoir of this precursor of the antibiotic peptide LL-37.     

Canine cathelicidin (K9CATH): gene cloning, expression, and biochemical activity of a novel pro-myeloid antimicrobial peptide

Cathelicidins, a group of cationic peptides found in leukocytes and epithelial cells, play a central role in the early innate immune defense against infection. Although these host defense peptides have been reported in several mammalian species, including primates, no cathelicidins have been identified in carnivores. Here we report the cloning, tissue expression and biological activity of a novel canine cathelicidin (K9CATH). The full-length cDNA sequence of K9CATH encodes a predicted 172 amino acid pre-propeptide that is 60–70% similar to other mammalian cathelicidins. Mass spectrometry analysis confirmed that the 38 aa mature K9CATH peptide was present in neutrophil granule contents. Synthetic K9CATH displayed broad antimicrobial activity against Gram-positive bacteria (Listeria monocytogenes, and Staphylococcus aureus; MICs (minimal inhibitory concentrations) 0.5 and 50 μM, respectively), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella serotype Typhimurium, Pseudomonas aeruginosa, Proteus mirabilis; MICs 1.25 μM, Salmonella serotype Enteritidis; MIC 0.5 μM, and Neisseria gonorrhoeae; MIC 0.06 μM), and yeast (Candida albicans; MIC 12.5–50 μM). K9CATH demonstrated high antimicrobial activity against Ureaplasma canigenitalium, and lower activity against Ureaplasma urealyticum (MIC 0.06 and 50 μM, respectively). Similar to its ovine congener SMAP-29, K9CATH possesses salt-independent antimicrobial activity and LPS binding capacity. K9CATH displayed minimal hemolytic activity against human, dog and chicken erythrocytes. The potency and broad antimicrobial activity of K9CATH suggest that this peptide may act as a fundamental contributor to the innate immune responses in this carnivore species     

Pulmonary defense and the human cathelicidin hCAP-18/LL-37

Antimicrobial peptides form an important component of the innate immune system. The cathelicidin family, a key member of the antimicrobial peptide defenses, has been highly conserved throughout evolution. Though widespread in mammals, there is currently only one identified human example, hCAP-18/LL-37. The cathelicidins have been found to have multiple functions, in addition to their known antimicrobial and lipopolysaccharide-neutralizing effects. As a result, they profoundly affect both innate and adaptive immunity. Currently, antimicrobial peptides are being evaluated as therapeutic drugs in disease states as diverse as oral mucositis, cystic fibrosis, and septic shock. One such peptide, the cathelicidin hCAP-18/LL-37, is reviewed in detail in the context of its role in lung physiology and defense.     

Effects of F/G-actin ratio and actin turn-over rate on NADPH oxidase activity in microglia

Background

Egg white must provide nutrients and protection to the developing avian embryo. One way in which this is achieved is an arsenal of antimicrobial proteins and peptides which are essentially extensions of the innate immune system. Gallin is a recently identified member of a family of peptides that are found in egg white. The function of this peptide family has not been identified and they are potentially antimicrobial.

Results

We have confirmed that there are at least 3 forms of the gallin gene in the chicken genome in 3 separate lines of chicken, all the forms are expressed in the tubular cells of the magnum region of the oviduct, consistent with its presence in egg white. mRNA expression levels are in the order 10,000 times greater in the magnum than the shell gland. The conservation between the multiple forms of gallin in the chicken genome compared with the conservation between gallin and other avian gallin like peptides, suggests that the gene duplication has occurred relatively recently in the chicken lineage. The gallin peptide family contains a six cysteine motif (C-X₅-C-X₃-C-X₁₁-C-X₃-C-C) found in all defensins, and is most closely related to avian beta-defensins, although the cysteine spacing differs. Further support for the classification comes from the presence of a glycine at position 10 in the 41 amino acid peptide. Recombinant gallin inhibited the growth of Escherischia coli (E. coli) at a concentration of 0.25 μM confirming it as part of the antimicrobial innate immune system in avian species.

Conclusions

The relatively recent evolution of multiple forms of a member of a new defensin related group of peptides that we have termed ovodefensins, may be an adaptation to increase expression or the first steps in divergent evolution of the gene in chickens. The potent antimicrobial activity of the peptide against E. coli increases our understanding of the antimicrobial strategies of the avian innate immune system particularly those of the egg white and the evolution of the defensin family. The potential of this peptide and others in the family can now be investigated in a number of novel antimicrobial roles.     

Sodium butyrate inhibits Staphylococcus aureus internalization in bovine mammary epithelial cells and induces the expression of antimicrobial peptide genes

A distinctive feature of bovine milk fat is the presence of butyrate, molecule with recognized antimicrobial and antiinflammatory properties. Bovine mastitis is a pathology characterized by inflammatory and infectious processes; however, the role of sodium butyrate on Staphylococcus aureus infection in mammary epithelium has not been studied. In this work we assess the role of sodium butyrate on the invasion of bovine mammary epithelial cells (bMEC) by S. aureus responsible of mastitis and on the expression of antimicrobial peptide genes. Our data show that sodium butyrate (0.25–0.5 mM) reduces ∼50% the internalization of S. aureus (ATCC 27543) into bMEC. By RT-PCR analysis, we showed that sodium butyrate is able to up-regulate the expression of tracheal antimicrobial peptide (TAP), β-defensin and inducible nitric oxide synthase (iNOS) mRNAs, as well as nitric oxide production. Also, sodium butyrate and infection increased acetylation of histone H3 in bMEC. These results indicate that sodium butyrate could be effective to modulate innate immune gene expression in mammary gland that leads to a better defense against bacterial infection. To our knowledge, this is the first report that shows a role of sodium butyrate during the internalization of S. aureus into bMEC.