【作者】 宁亚蕾；

【导师】 邹全明；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2009， 博士

【摘要】 缺乏高效、稳定、低成本、易纯化的重组蛋白表达系统是目前基因工程药物/疫苗研发、生产和相关生物技术的瓶颈之一。目前的表达载体几乎均以胞浆或周质间隙形式表达重组目的蛋白,存在种种缺陷,给下游工作带来重重困难。例如高表达的重组目的蛋白常常在胞内形成包涵体,生物活性不佳,复性困难;周质间隙表达虽可增加重组蛋白的可溶性,避免包涵体复性的繁琐程序,但其表达量有限且对重组蛋白的分子大小和结构有所限制,尤其不适合大规模生产。而胞外分泌型表达是提高重组蛋白有效产率和生物利用度更为理想的途径,因为胞外环境有利于蛋白的折叠和稳定,从而显著增加重组蛋白活性;同时胞外环境蛋白酶及宿主蛋白的干扰相对最少,重组蛋白更稳定,更易于纯化;此外还能够减轻对宿主菌及重组蛋白本身的毒性,并能持续分泌表达。实现重组蛋白胞外递送的一个有效途径是移植利用细菌的天然分泌系统,该途径与非特异的渗漏表达不同,具有高效、特异和高度可控的优点。因此本课题旨在将具有胞外分泌功能的新月柄杆菌RsaA分泌系统移植到原核表达最常用的工程菌E. coli中,使其成为重组蛋白胞外递送的工具,以发展具有自主知识产权的高效通用胞外分泌表达系统pQABPS。但新月柄杆菌本身的调控元件不能为E. coli所识别,简单地将RsaA分泌系统直接移植到E. coli中是不能重现胞外分泌功能的,需要依赖于外源基因表达调控序列的作用,使该系统多个功能基因在E. coli中以合理水平组成型表达。因此本课题拟通过基因操作,获得RsaA分泌系统功能基因rsaD、rsaE、rsaFa、rsaFb(RsaFa和RsaFb共同承担RsaF功能)及C-端信号序列rsaAs,并配合有效的外源调控序列(包括终止子、启动子、翻译增强序列及RBS等),构建到pQE30骨架载体上,得到原核胞外分泌型表达载体系统pQABPS,通过表达四个大小、来源不同及功能明确的外源重组蛋白GFP、EspA、UreB和hIL-24,检测其表达量与活性,验证其通用性及质粒稳定性,并在调控元件强度、宿主菌及分泌功能基因组织形式等方面对该系统进行优化。本课题完成了以下几个方面的工作:1.外源基因表达调控序列的筛选。首先克隆了六个基因表达调控序列rrnBT1(核糖体终止子)、强组成型T5启动子PTS及其突变体PTW、中等强度组成型β-内酰胺酶启动子PB及包含其本身SD序列的PBS,以及翻译增强序列Epsilon(T7噬菌体gene-10翻译增强子序列)及SD序列(简写为ESD)。然后观察在不同的宿主菌E. coli JM109和E. coli M15中,不同的调控序列组合调控报告基因GFP的表达情况,结果表明,rrnBT1-PTS-ESD和rrnBT1-PB-ESD两个组合效果较好。随后验证了它们对RsaA系统各单个分泌功能基因rsaD、rsaE、rsaFa和rsaFb的表达调控作用,结果表明它们起到了预期的作用,可以用于下一步的载体构建。2.胞外分泌表达载体pQABPS的构建及鉴定1)克隆了四个5’端带ESD序列的RsaA分泌功能基因rsaD、rsaE、rsaFa和rsaFb,利用同尾酶将它们连接起来,得到ESDDEFab片段,与第一部分筛选出的外源调控序列组合rrnBT1-PTS/rrnBT1-PTB及RsaA分泌系统C端信号序列rsaAs一起克隆至pQE30骨架载体,得到本课题的目的载体pQABPS。2)用该载体表达报告分子GFP,结果表明GFP能够被分泌至胞外培养基中,分泌量为1.0mg/L;同时在转录和翻译两个水平面检测到了分泌功能蛋白的组成型表达,结合Western blotting结果,证明GFP的胞外运输是通过特异的RsaA转运装置而非渗漏表达。3)质粒稳定性检测结果显示,在有选择压力存在的情况下,pQABPS质粒在M15中能够稳定存在60代,60代之后丢失率稍增加,总体来说稳定性良好。3.胞外分泌表达载体pQABPS的优化及应用初探1)克隆了三个大小、来源不同的外源蛋白EspA、UreB和hIL-24验证pQABPS/M15表达系统的通用性,结果表明,它们均被成功分泌至胞外培养基中。2)用ELISA试剂盒定量检测了培养上清中重组hIL-24的表达量,为336.5μg/L,随后直接用培养上清(对照为M15空菌培养上清)进行了体外诱导肿瘤细胞凋亡的实验,证实了上清中的hIL-24具有体外诱导肿瘤细胞凋亡的活性,且对正常细胞没有影响。说明pQABPS/M15系统分泌表达有利于外源蛋白的生物学活性。3)将分别载有四个外源蛋白GFP、EspA、UreB和hIL-24的pQABPS质粒转入E. coli XL1-Blue宿主菌中,观察不同宿主菌对分泌效果的影响,结果表明诱导表达后同样能在培养上清中检测到目的蛋白的表达。4)构建了缺失rsaFb基因(仅保留rsaD、rsaE和rsaFa基因)的质粒载体pQG2ABPS(-)、pQA2ABPS(-)、pQU2ABPS(-)和pQI2ABPS(-),将它们转入E. coli M15中,观察不同的分泌功能基因组织形式对分泌效果的影响,结果表明诱导表达后同样能在培养上清中检测到目的蛋白的表达,表达量没有明显变化。综上所述,本课题构建了基于新月柄杆菌RsaA系统的胞外分泌表达载体pQABPS。该载体具有一定的通用性,能够将外源蛋白特异地转运至胞外培养基中,且有利于生物学活性的保留。该质粒载体在宿主菌E. coli M15中具有良好的遗传稳定性。更多还原

【Abstract】 Obtaining soluble proteins often constitutes a bottleneck in the production of recombinant proteins for drug developments, structural studies and proteomics. This can be accounted for, at least in part, the lack of efficient secretion systems capable of large-scale exportation of recombinant proteins. Secretory expression of recombinant proteins in Escherichia coli has exhibited several advantages over intracellular production, including: 1) to facilitate the downstream process; 2) to enhance the biological activity; 3) to increase the stability and solubility of recombinant proteins. Among the five major secretion systems (type I, II, III, IV, and V) that can export protein into the extracellular space. Type I (ATP-binding cassette) secretion system featuring a number of striking features has been recognized to be an attractive candidate for development of extracellular expression systems of recombinant proteins.In E. coli, the a-haemolysin (HlyA) transporter is a representative member of the type I transporter family. Indeed, it has been utilized extensively. Despite many advantages, the HlyA secretion pathway has a number of drawbacks. Probably, there is not sufficient room for further improvement of HlyA-based secretion systems, demanding the exploration of more efficient and less restrictive secretion systems for recombinant protein exportation.RsaA (regular surface array protein) secretion system in Caulobacter crescentus, which mediates the secretion of surface layer protein (SLP) is considered as a promising candidate for development of improved recombinant protein secretory delivery systems. Theoretically, as long as the four secretion element genes rsaD, rsaE, rsaFa and rsaFb were cloned and expressed, the heterologous proteins fused to RsaAs, the rsaA C-terminal secretory signal, will be secreted to the extracellular medium. However, the regulatory elements of C. crescentus couldn’t be recognized by the host E. coli. So some other regulatory elements that can work in E. coli should be involved.In this work, we cloned a series of genes encoding exporter proteins rsaD, rsaE, rsaFa, rsaFb, the C-terminal signal sequence rsaAs and three other exogenous regulatory components respectively. These genes and the foreign regulatory components were directionally inserted into pQE30 vector, resulting in the plasmid, termed as pQABPS. Then four heterologous proteins (GFP, EspA UreB and hIL24) from different origins and of different sizes were fused to the C-terminal signal for evaluation of the extracellular secretion capacity of the newborn plasmid pQABPS.The study has been involved in the following aspects:1. Selection and determination of exogenous regulatory componentsSix gene expression regulatory components—strong terminator rrnBT1, strong constitutive T5 promotor PTS and its mutant PTW, moderate constitutiveβ-lactamase promoter PB and PBS (its SD sequence was included), Epsilon (UUAACUUUA, T7 phage gene 10 translation enhancer), along with Shine-Dalgarno (SD) consensus sequence AAGGAG (abbreviated as ESD), were cloned. SDS-PAGE analysis showed that the reporter protein GFP was constitutively expressed under the regulation of them. Two optimized group rrnBT1-PTS-ESD and rrnBT1-PB-ESD were selected to regulate the expression of four secretion element genes.2. Development and evaluation of the extracellular secretion vector pQABPS1) Four Caulobacter crescentus RsaA secretion elements rsaD, rsaE, rsaFa and rsaFb and three exogenous regulatory components were directionally inserted into pQE30 vector, generating the interested plasmid designated pQABPS.2) The reporter protein GFP were subjected to expression assays using this newly-developed expression system, pQABPS/E. coli M15. The hybrid protein in the culture supernatant was detected by SDS-PAGE and Western bloting. The secretion amount was about 1.0mg/L. To test whether the target protein was secreted through the special RsaA transporter, expression of four structural proteins RsaD, RsaE, RsaFa and RsaFb was tested by RT-PCR and SDS-PAGE. As a result, the GFP hybrid protein was exported from the E. coli cells in an RsaA secretion system dependent manner, but not a nonspecific leakage after long-time cultivation.3)The stability of the pABPS plasmid in E. coli M15 was analysed. It showed that the plasmid had good genetic stability in M15 with selective pressure.3. Application and optimization of the pQABPS vector1) Three other heterologous proteins, EspA (E. coli secreted protein), UreB (H. Pylori urase subunit B) and hIL-24 (human Interleukin-24) -with different sizes and from different origin were selected to evaluate the extracellular secretion capability of the newly-developed pQABPS vector system. Results of Western blotting analysis revealed that they were efficiently secreted into culture supernatant.2) Extracellular expression product of recombinant hIL-24 was identified by ELISA. It showed that the secretion amount of hIL-24 was 336.5μg/L. The biological activity of inducing tumor cells apoptosis of recombinant hIL-24 was detected by MTT and morphology overview. It showed that the recombinant hIL-24 in the culture supernatant of M15 induced apoptosis of tumor cells in vitro, while and no effect on normal cells. It revealed that the hybrid hIL-24 expressed extracelluarly remained biological activity.3) The new host E. coli XL1-Blue harbouring pQABPS can also export the four heterologous proteins GFP, EspA, UreB and hL-24 to the outer space.4) Four plasmids lacking of rsaFb gene pQG2ABPS(-), pQA2ABPS(-), pQU2ABPS(-) and pQI2ABPS(-) were constructed and transformed to E. coli M15. Western blotting analysis revealed that the four heterologous proteins GFP, EspA, UreB and hL-24 were all secreted to the culture supernatant. There were no obvious change of secretion capacity.In conclusion, novel extracellular expression system, pQABPS/E. coli (M15), carrying Caulobacter crescentus RsaA secretion elements was developed. Caulobacter crescentus RsaA secretion elements and three other exogenous regulatory components were directionally inserted into pQE30 vector, generating the interested plasmid designated pQABPS. Four heterologous genes (gfp, espA, ureB and il24) were subjected to expression assays using this newly-developed expression system, pQABPS/E. coli (M15). Western blot analysis and bioactivity tests revealed that they were efficiently secreted into culture supernatant and remained biological activity, implying that pQABPS fulfills the capability of extracellular delivering bioactive foreign proteins in E. coli (M15). Taken together, the expression system of pQABPS/E. coli (M15) can function as another useful tool for extracellular secretary expression of exogenous proteins.更多还原