Study on Vaccine of Recombinant Peptide and DNA Based on Avian Influenza virus M2 and Mucosal Immunization

禽流感病毒（Avianinfluenzavirus,AIV）能感染几乎所有的野生及饲养禽类，引起禽类尤其是饲养禽类的死亡，AIV的流行给养禽业造成了严重的经济损失。1997年在香港发现H5N1亚型禽流感病毒突破生物屏障直接感染人，使人们认识到AIV对于人类公共卫生的重要性。疫苗接种是预防和控制AIV流行的主要措施之一，但AIV突变频率高、亚型多，给疫苗的研制带来困难。AIV的M2蛋白是病毒囊膜上的蛋白之一，具有亚型间高度的保守性，是理想的交叉保护性疫苗的候选抗原。AIV主要是经过动物呼吸道等粘膜系统传染，具有诱导机体粘膜和系统应答能力的疫苗对于禽流感的防治具有重要的意义。大肠杆菌不耐热肠毒素（...Abstract Avian influenza virus (AIV) can infect almost all wild bird and domestic poultries and especially lead to the death of poultry. Severe economic loss for the poultry industry was brought because of the AIV prevalence. In 1997, H5N1 subtype of AIV was isolated from flu patient in Hong Kong, the subtype ultimately caused several patients dead, and it was the first time reported that H5N1 AI...学位：理学博士院系专业：生命科学学院生物化学与生物技术系_细胞生物学学号：B20042601

Expression and Construction of Yeast Expression Vector of LBM2eHBc+ gene in P.pastoris

禽流感病毒一直是家禽和家畜健康养殖的巨大威胁,之前的研究表明,原核系统中表达的lTb和M2EHbC+融合基因的产物能有效诱导免疫动物对禽流感病毒M2E多肽产生特异性的粘膜免疫应答.酵母作为生物反应器应用于生物制品生产具有独特的优点.本研究构建了PPIC9k-lbM2EHbC+酵母表达载体并对酵母gS115进行了遗传转化,甲醇诱导表达结果表明lbM2EHbC+融合基因在酵母细胞中得到表达,表达出的融合蛋白能够被M2抗体识别.Avian influenza virus(AIV) has been a constant threat to the healthy development of livestock and poultry breeding industry.Previous research has shown that the products of LTB and M2eHBc+ fused gene expressed in prokaryotic cells can effectively induce mice mucosal immune responses against M2e epitope.P.pastoris yeast has a unique advantage as a bioreactor used in the production of biological products.LBM2eHBc+ fused gene fragment was obtained by PCR,then inserted into pPIC9K plasmid which contains methanol promoter and yeast signal peptide to construct yeast expression vector pPIC9K-LBM2eHBc+.The recombinant vector was transformed into P.pastoris GS115.The methanol induction result has indicated that the LBM2eHBc+ fusion gene can be efficiently expressed in GS115 cell and the expressing protein has the specific binding activity to M2 antibody by Western blotting analysis..福建省科技厅重大项目子课题(2006NZ0003-2);福建省教育厅A类科技项目(JA09168

Studies on Construction of Plant Expression Vector of Recombinant LBM2eHBc+ Gene and Tomato Transformation

禽流感病毒一直是家禽和家畜健康养殖的巨大威胁,甚至威胁着人类的健康,人们也一直在研究各种形式的疫苗来应对禽流感病毒的威胁.番茄作为生物反应器应用于动物疫苗生产具有独特的优点,本研究优化了番茄子叶的再生体系,同时构建了lbM2EHbC+融合基因的植物表达载体并对番茄进行了遗传转化.结果表明在玉米素(zT)浓度为0.5 Mg/l时番茄子叶外植体的芽再生率达到93.33%;测序结果证明植物表达载体PbI121-lbM2EHbC+构建成功;利用农杆菌介导的转化方法获得再生抗性苗34株,经PCr检测,阳性率为75.0%,本研究结果为进一步开发禽流感口服疫苗奠定了基础.Avian influenza virus(AIV) infect almost all wild bird and domestic poultries and then decimate poultries,even the threat of AIV overhangs human.It also has been researching a variety of vaccines to combat the AIV threat.Tomato as a bioreactor used in animal vaccine production has a unique advantage.This study optimized the regeneration system of tomato cotyledon,while construct LBM2eHBc + gene plant expression vector,and then carried out the genetic transformation of tomato.The results show that the bud regeneration rates of tomato reach the highest 93.33% when MS medium supplemented 0.5 mg/L Zt+ 1 mg/L IAA;sequence results show that plant expression vector pBI121-LBM2eHBc + constructed successfully.A total of 34 putative transgenic tomato plants were obtained by using of Agrobacterium-mediated transformation method,and the positive rate was 75.0% by PCR detection.The results laid a foundation for the further development of oral AIV vaccines.福建省科技厅重大项目子课题(2006NZ0003-2);福建省教育厅A类科技项目(JA09168

The Effects of Different Lights and Gibberellin on Establishment of Parasitism between Dodder and Its Hosts

首次使用lEd作为光源,研究不同光照条件及gA3对菟丝子(CuSCuTA SPP.)弯钩打开、缠绕发生与吸器形成的影响。结果表明,光照信号作为一个必要条件参与了菟丝子对寄主的识别及缠绕发生的调控,而化学信号可能起到一定的促进作用;gA3参与了对菟丝子缠绕发生的调控,但对弯钩打开没有明显的作用。除了典型的光敏色素作用外,还有另一类光反应(HEr)参与了上述过程,这类光反应可由879 nM远红光引发,证明菟丝子存在HEr,还有Pfr向Pr的暗转化过程,在缠绕发生过程中光敏色素和隐花色素发生相互作用。The effects of different light treatments and GA3 on hook opening,twining and parasitism of Cuscuta australis were studied using LED as light sources for the first time.The results showed that light was a necessary factor for dodders to parasitize the hosts successfully and chemical signals might facilitate host recognition and twining.GA3 involved in controlling twining response,but no distinct effect on hook opening.Furthermore,besides typical(phytochrome reaction,another photoreaction called HER was involved in these processes,which could be caused by 879 nm far red light.So it demonstrated directly that there was not only HER in dodders,but also dark conversion from Pfr to Pr,and there were mutual interaction of phytochromes and cryptochromes in twining.教育部重点项目(101102)资

Study on Tomato Transformation with Human Keratinocyte Growth Factor-1

人角质细胞生长因子在组织的损伤修复中起着重要的作用.以番茄子叶为外植体,通过根癌农杆菌介导法,将人kgf-1导入番茄中,共获得12株独立的抗性转化植株,PCr和dnA斑点杂交结果表明:kgf-1基因整合入番茄基因组中,为获得植物源表达的kgf-1蛋白打下了基础.Keratinocyte growth factor plays an important role in the tissue damage repair.Using sterile tomato cotyledons as explants,the human KGF-1 gene had been introduced into the tomato genome by Agrobacterium-mediated transformation method.As a result,a total of 12 independent putative transgenic tomato plantlets were obtained.The results of PCR and DNA dot-blot hybridization showed that KGF-1 gene was integrated into the tomato genome.This work has laid a foundation for further studies on the development of KGF-1 proteins expressed in tomatoes.福建省自然科学基金(2010J01240

Expression of Modified H5N1 Avian Influenza Virus M2 Gene and the Recombinant M2 Protein Immunogenicity Assay

禽流感的发生不仅会造成禽类的大量死亡,而且也严重地威胁着人类健康,接种疫苗是控制禽流感发生的主要措施之一.M2基因的保守性使其成为基因工程亚单位疫苗的目标抗原.本研究将M2基因的跨膜区删除后,构建原核表达载体PET32A-△M 2,IPTg诱导后,收获的细菌总蛋白SdS-PAgE电泳结果表明修饰的M2基因在原核表达系统中得到高效表达,表达蛋白以可溶性形式存在,WESTErn杂交和动物免疫结果表明重组蛋白具有抗原性和免疫原性.本研究结果为利用M2重组蛋白开发具有交叉保护作用的禽流感疫苗奠定了基础.The present vaccination is one of main strategy for control of avian influenza virus that critically threatened human health.Recent investigations of subunit component vaccine focused on M2 gene because M2 gene possessed a highly structurally conserved property among influenza viral strain.In this study,the recombinants prokaryotic expression plasmid pET32a-△ M2 harboring the deletion of the transmembrane segment of M2 gene was constructed.SDS-PAGE electrophoresis showed that modified M2 gene was highly expressed as fusion protein in a soluble form after genetic modified E.coli BL21(DE3) bacteria were induced with IPTG.The fusion protein of M2 possessed antigenicity and immunogenicity properties by Western Blotting analysis of fusion protein with standard antibody against M2(H5N1) and mouse vaccination of purified fusion protein respectively.The results laid a foundation of further study on the development of cross-protection avian influenza vaccine.福建省科技厅重点项目(2009N0051);福建省教育厅A类科技项目(JA09168

A Convenient Method for Condon Adjustment by Using High-fidelity DNA Polymerase Amplification Property

在基因工程研究中,经常要涉及到对目的基因读码框进行调整的研究。本文报道了利用高保真DNA聚合酶扩增DNA后产生平末端的特性,及限制性内切酶对一重组的原核表达载体pET28a-HA进行读码框调整的研究,测序结果显示读码框按照预期设计正确调整,表明高保真DNA聚合酶可以用于DNA 3'凹陷端的补平,在DNA 3'凹陷端平滑化时多了一个有效的选择。The reading frame adjustment of target genes was often occurred in the study of gene engineering.The results of pET28-HA gene reading frame exactly adjustment showed high-fidelity DNA polymerase can be used generate blunt end from a DNA 5'-protruding end.The convenient method can be selected in the blunt of DNA 5'-protruding end

Recombinant Keratinocyte Growth Factor-1 Expression in Rice

人角质细胞生长因子(kErATInOCyTE grOWTH fACTOr,kgf)在组织的损伤修复中起着重要的作用.植物细胞生物反应器是一种成本低、安全性好的蛋白生产系统.本研究将人kgf-1经农杆菌介导法转入水稻中,共获得38株抗性再生植株.SOuTHErn杂交和rT-PCr结果表明kgf-1基因整合入水稻基因组中且部分转化株中kgf-1基因得到转录.WESTErn blOTTIng检测结果显示有两株转化水稻中能够表达kgf-1.Keratinocyte growth factor(KGF) plays an important role in the tissue damage repair.Plant cell bioreactor is a low cost and safe protein production system.Human KGF-1 gene was transformed into rice by Agrobacterium-mediated method and a total of 38 putative transgenic rice plants were obainted.The results of southern blotting and RT-PCR assay showed that KGF-1 gene was integrated into the rice genome and KGF-1 gene was transcribed in part of the putative transformed rices.Western blotting results showed that KGF-1 was expressed in two transgenic rices.福建省自然科学基金资助项目(2010J01240

Expressional Optimization of Prokaryotic Expression Vector of MS2 Phage and Assay of the Stability of Virus-like Particles

MS2噬菌体病毒样颗粒广泛用于构建rnA病毒检测中的阳性对照品.本文基于已构建的表达载体P CPHA,优化了表达载体在bl21(dE3)细胞中的表达条件.结果表明在lb培养基中,菌液Od600=0.9时,加入IPTg诱导6H,外壳蛋白的表达量达到最大,电镜下观察到表达出的产物呈现MS2噬菌体病毒颗粒结构,该病毒样颗粒在常温下至少可以保存60 d,64℃温度时30 MIn内结构保持稳定.Virus-like particle of MS2 bacteriophage was widely used as a kind of positive control in detection of RNA virus.Several expressional conditions of p CPHA vector that pack hemagglutinin(HA) gene segment of avian influenza virus were optimized inside E.coli BL21(DE3) cell.The results showed coat protein expression level reaches the maximum at the broth OD600 of 0.9,IPTG induction 6h and LB liquid medium.Expression product morphological structure was the same as MS2 bacteriophage virus particles under scanning electron microscope.The structure of virus-like particle was stable at normal temperature for at least 60 d and 64℃ for at least 30 min

Plant Regeneration in vitro of Cuscuta chinensis Lam.

选取萌发3～5d、长度3～5cm的中国菟丝子(Cuscuta chinensis Lam.)幼苗,将其分为上、中、下3部分并作为外植体进行离体培养与植株再生研究。结果表明,其上、中部片段更适宜愈伤组织诱导;诱导培养基以添加1mgL-1NAA和1mgL-1BA的MMS培养基效果最好,此培养基也可用于愈伤组织的继代培养,愈伤组织在上述培养基中已生长一年之久。分化培养基为添加1mgL-1BA的MMS培养基,平均每块愈伤组织可以产生2.8株植株。An efficient method of callus induction and plant regeneration of Cuscuta chinensis was established.The seedlings germinated for 3~5 d and about 3~5 cm length were selected as explants,and then divided into three parts,upper,middle and lower.Callus were inducted from upper or middle parts of seedlings on a modified Murashige and Skoog(MMS)medium supplemented with 1 mg L-1 NAA and 1 mg L-1 BA.The calli have been subcultured on such medium for over a year.Shoot regeneration from callus was achieved on MMS medium containing 1 mg L-1 BA and could obtain 2.8 shoots per callus