腺病毒介导的人乳铁蛋白基因在兔和羊乳腺中高效表达的研究
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摘要
动物乳腺被认为是生产基因工程蛋白药物的最佳场所之一,外源目标基因通过乳腺上皮细胞表达,自然分泌到动物乳汁中,从而获得所需目标蛋白。因此,研究者们在研发如何利用动物乳腺生物反应器生产转基因蛋白药物方面进行了大量探索。其主要步骤包含:(1)构建乳腺特异表达载体,在构建乳腺表达的外源基因时,外源基因在乳腺特异性表达需要乳蛋白基因的一个启动子和调控区,即要有一个引导泌乳期乳蛋白基因表达的序列,这样才能将外源基因置于乳腺特异性调节序列控制之下,使其在乳腺中表达,再通过回收乳汁得到目标蛋白。(2)通过各种转基因方法将乳腺特表达载体导入原核期胚胎或体细胞,获得重组胚胎或转基因细胞;(3)将筛选正确的转基因胚胎或转基因体细胞,通过胚胎移植或核移植技术获得转基因动物个体;(4)将转基因动物个体进一步繁育,筛选检测转基因动物后代乳汁中目标蛋白基因的表达,获得稳定遗传目标基因的转基因动物。然而,现存的技术体系仍存在不可避免的缺陷:(1)需要复杂的技术程序操作,导致转基因结果不稳定。(2)该方法在大动物上转基因成功率极低,造成生产成本居高不下。(3)由于转入的外源目标基因的位置效应,使得目标基因表达水平很低。(4)外源基因在动物体内的异源表达往往给动物健康造成伤害,影响基因工程蛋白药物的生产。目前,国际上很难获得乳腺表达转基因动物。
针对上述技术体系缺陷,本研究探索了以腺病毒为载体,直接导入动物乳腺表达生产转基因蛋白药物的方法。目标基因在动物乳腺上皮细胞中表达,并分泌到乳汁中,从而在动物乳汁中获得重组蛋白药物。其优势在于:(1)不需要构建乳腺特异表达载体,从而简化了转基因蛋白药物生产程序;(2)以腺病毒为载体直接导入乳腺,可获得高表达的目的蛋白药物;(3)乳腺组织可以对人体蛋白质进行正确的修饰和转录后加工,蛋白药物产品的生物学活性接近天然产品;(4)目标蛋白药物在乳腺中高水平短期表达,无需对动物进行长期维持培育,可降低生产成本。
腺病毒载体以其高效转移并表达目标基因和安全性高诸多优点被应用于基因转移之中。但是,国内外有关腺病毒载体的应用一直侧重于基因治疗方面。目前国际上有关腺病毒基因治疗的临床实例应用已上千种,而国内近3年来也已批准了3个有关腺病毒载体应用于人类基因治疗的基因工程药物。但是,迄今为止,腺病毒载体在重组药物蛋白生产方面至今未见到相关应用报道。为此,本研究以人乳铁蛋白重组腺病毒为载体,进行了乳腺表达生产人乳铁蛋白的试验研究,以期探索出高效、简便的转基因蛋白药物生产方法,从而为转基因蛋白药物生产提供新的途径。
本研究共分为五个部分,第一、二部分进行了人乳铁蛋白基因表达载体的构建及人乳铁蛋白cDNA的体外细胞表达,为后面目标基因在动物体内的表达奠定了基础。第三、四部分主要对2种腺病毒载体的构建方法进行了探讨,获得了人乳铁蛋白重组腺病毒高效制备的方法,为进一步利用腺病毒为载体进行乳腺表达奠定了基础。第五部分利用构建成功的重组人乳铁蛋白腺病毒载体,乳腺表达生产人乳铁蛋白蛋白,探讨腺病毒为载体生产转基因蛋白药物的可行性、稳定性及高效性。
第一部分人乳铁蛋白cDNA序列的克隆及真核表达载体的构建实验中,首先从人乳腺组织分离提取得到总RNA,利用RT-PCR方法,直接获得人乳铁蛋白的全长cDNA序列,经DNA测序分析,所得hLTF cDNA与GeneBank中一致。并对克隆的人乳铁蛋白基因的编码氨基酸序列进行分析,结果表明,所得人乳铁蛋白基因序列编码的氨基酸与已发表的人乳铁蛋白编码序列完全一致。以pcDNA3.1、pIRES、pDC316为基础质粒,构建了含有人乳铁蛋白基因的重组4种真核表达载体p3LF、pDCLF、pLG和p3LG。其中,真核表达载体p3LF和pDCLF,含有新霉素抗性筛选标记,可进行转染哺乳动物细胞的抗性筛选,真核表达载体pLG和p3LG,内含有GPF报告基因,可对目标蛋白基因的表达进行实时观测,极大提高了目标蛋白基因检测效率,为后面人乳铁蛋白的表达研究奠定了基础。第二部分人乳铁蛋白cDNA的细胞表达实验中,将上述构建的人乳铁蛋白真核表达载体p3LF、pDCLF、pLG和p3LG分别在COS-7、CHO、和HEK293细胞3种不同细胞株中进行了蛋白表达实验。比较不同真核表达载体在不同哺乳细胞株中的表达效率方法。将其转染进细胞后,CHO和HEK293细胞经G418抗性筛选,获得阳性克隆细胞,并克隆化培养,RT-PCR和Western blotting分析显示,培养上清液中分别有较高的hLTF mRNA和蛋白表达,而COS-7细胞未经G418筛选,直接于转染后48h开始检测细胞培养上清,也获得了蛋白表达。体外抑菌实验证实,重组人乳铁蛋白具有较强的抗菌活性。初步建立了体外真核细胞蛋白表达体系,为后面人乳铁蛋白腺病毒载体构建研究奠定了基础。第三部分细菌内高效制备重组人乳铁蛋白腺病毒Ad-hLTF载体的研究,将体外真核表达验证正确的人乳铁蛋白基因亚克隆至穿梭质粒pShuttle-cmv中,酶切鉴定后所得重组质粒pShuttle-hLTF,经PmeI酶切线性化后,与腺病毒骨架质粒pAdeasy共转化大肠杆菌BJ5183,所获阳性菌落酶切鉴定正确后,获得的重组人乳铁蛋白腺病毒载体Ad-hLTF,经PacI消化后,将大片段纯化后转染HEK293细胞,包装获得重组人乳铁蛋白腺病毒。PCR鉴定病毒上清含有目标蛋白基因hLTF,Western blotting检测到感染细胞上清中含有人乳铁蛋白的表达。结果表明,利用细菌内同源重组法制备的Ad-hLTF重组腺病毒载体是一种方便、高效的方法,所制备的重组腺病毒在体外能有效表达具有生物活性的hLTF,为后面体内实验研究奠定了基础。
第四部分细胞内高效制备重组人乳铁蛋白腺病毒Ad-hLTF载体的研究,为探索利用真核细胞内同源重组生产腺病毒方法,该方法基本原理是通过Cre-loxP或FLP-frt重组酶,使转染进293细胞的Shuttle质粒和辅助大质粒发生定点重组,产生重组腺病毒。得到的重组病毒是E1/E3缺失的复制缺型腺病毒。将目标蛋白基因hLTF克隆至质粒pDC315,该质粒MCS两侧包含有Cre-loxP位点,获得的重组质粒pDC311-hLTF,将其与辅助腺病毒质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞,获得真核细胞内重组包装重组腺病毒Ad-hLTF,PCR鉴定病毒上清含有目标蛋白基因hLTF,Western blotting检测到感染细胞上清中含有人乳铁蛋白的表达,体外抑菌实验结果表明,重组人乳铁蛋白具有较强的抗菌活性。结果表明,利用真核细胞内同源重组法制备的Ad-hLTF重组腺病毒载体是一种更为简便的方法,省去了细菌重组构建大质粒的筛选鉴定过程,所制备的重组腺病毒在体外能有效表达具有生物活性的hLTF。
第五部分重组腺病毒Ad-hLTF在兔和羊乳腺中高效表达的研究,将上述构建正确的重组hLTF腺病毒载体,在HEK293细胞中经过4轮扩增,所获病毒经进一步纯化后,最终获得病毒滴度1011-1012PFU/ml。通过将重组hLTF腺病毒在兔和羊的乳腺中的体内表达实验,48 h后分别检测到了兔和羊奶中的hLTF的高表达。结果表明,所构建的重组hLTF腺病毒载体在兔和羊乳腺中均获得了高效表达,Western blotting检测病毒感染后2-10 d的兔和羊的乳汁,可检测到人乳铁蛋白的表达,从Western blotting人乳铁蛋白的特异条带结果分析,兔乳汁中人乳铁蛋白的表达量高于羊乳汁中hLTF的表达量。该体系尚待进一步改进和不断完善。下一步将继续探索腺病毒为载体在羊乳腺中的高效表达。以此,建立腺病毒为载体,乳腺表达生产转基因蛋白药物的技术体系。结论:(1)应用RT-PCR方法直接从人乳腺组织中扩增获得人乳铁蛋白cDNA全序列;(2)构建了4种不同的人乳铁蛋白真核表达载体;(3)获得了人乳铁蛋白cDNA在细胞的体外表达;(4)建立了细菌内同源重组高效制备人乳铁蛋白腺病毒载体的方法,并获得了表达有生物活性的人乳铁蛋白腺病毒载体;(5)建立了细胞内同源重组高效制备人乳铁蛋白腺病毒载体的方法,并获得了表达有生物活性的人乳铁蛋白腺病毒载体;(6)获得了重组人乳铁蛋白腺病毒在兔和羊乳腺中的高效表达。
本研究首次以腺病毒为载体,在动物乳腺中,获得了重组人乳铁蛋白的高效表达,为重组人乳铁蛋白的生产奠定了基础。该方法进一步拓展了腺病毒载体的应用,是乳腺生物反应器原理生产转基因蛋白药物产品的新应用。该方法无论在转基因技术理论领域,还是在基因工程药物生产方面,均具有其创造性和实用性。
The mammary gland has been identified as a suitable alternative to produce biopharmaceutical and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk from which the purification process is relatively simple. So, transgenesis has been extensively used for the genetic modification of farm animals to produce human recombinant proteins in milk. This system concluding four steps:(i)First,construct the recombinant expression vectors and special sequences are specific for mammary glad expression; But it is known that this control system is not absolute, allowing for transgene expression founders might die even in the fetal stage, where only the animals in which the expression levels were very low were born. (ii) Through some transgene methods and get the recombinant embryo or cells. (iii) Embryo transfer and nuclear transfer and got the transgene animal ;(vi) after breeding the transgene animals, which to be detected for expression target gene.
However, drawbacks in this technology include (i) the technically difficult procedure required, its low efficiency in produce transgenic animals;(ii) especially its inefficiency when applied to large livestock, (iii)the length of time between incorporating foreign DNA and harvesting the exogenous protein,(vi)and the health risk for animals due to the ectopic expression of transgenes. The direct in vivo transfection of the mammary gland has been proposed as a faster and more inexpensive alternative to target the expression of a heterologous gene to the secretory mammary epithelial cells. Although these expression levels may be enough for certain basic studies, they must be improved when large amounts of recombinant proteins are required.
Build on above traditionally technology system shortcomings,this paper reported a strategy to produce transgenic recombinant protein in the milk of animal mammary gland, such as rabbits and goats. Adenoviral vectors are able to successfully infect the mammary glandular epithelium and to promote the secretion of high amounts of a recombinant protein in the milk of animals. This system has some merits (i) the direct transduction of the mammary gland with adenoviral vectors could be a very useful tool to study the powerfulness of mammary-specific promoters avoiding variables associated to the position effects or to the copy numbers. The use of constitutive promoters, undoubtedly simplifies vector design, however this does not preclude the use of tissue specific promoters for special applications. (ii) It is possible to obtain high expression levels of a foreign protein in milk. (iii) The mammary gland can produce an in vivo biologically active recombinant protein. (vi) The secretion of the recombinant human lactoferrin in the milk is not permanence expression, so have low products cost.
Members of the adenovirus family infect a great variety of post-mitotic cells; even those associated with highly differentiated tissues such as skeletal muscle, lung, brain and heart. Since they deliver their genome to the nucleus and can replicate with high efficiency, they are prime candidates for the expression and delivery of therapeutic genes. They have a wide host-range and are currently divided into three genera with further subdivision into species (also termed subgenera or subgroups) A to F. Division of human stereotypes, based mainly on immunological criteria, has historically been the basis of classification
Recombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy. Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. The ability of adenovirus vectors to deliver and express genes at high yields, especially in vitro, however, to date, there has little reports have been used adenovirus for production of transgenic recombinant protein. The objective of our study is to investigate a new method by using recombinant adenovirus, which can produce the biopharmaceutical, and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk. The method may be used also to assess the functionality of gene constructs prior to the generation of a transgenic animal, and may be a viable approach to study the physical, chemical and biological characteristics of recombinant proteins expressed in milk of different species at the same time.
In order to establish a platform for the development of new biotechnological processes directed to the production of recombinants complex proteins in milk. We have carried out the studies on the cloning of objective gene and construction of mammalian expression vector and recombinant adenovirus containing hLTF cDNA, also, the rAd vectors direct transduction of the mammary gland. The main results are as following:
1. Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue, and then subcloned into pcDNA3.1 vector. hLTF cDNA sequence was determined, which consists of 2169bp. Comparison with five other hLTF cDNA sequences registered in GeneBank shows 99 % homology in DNA sequence. Four mammalian expression vectors were constructed, the pLG and p3LG vectors have GFP report gene and the incorporation of a GFP protein in this vector was designed to serve as a live marker for tracking infected cells or tissues in animal studies. The vectors p3LF and pDCLF use the neomycin resistance gene (Neor) to permit selection of transformed cells. The pLG plasmid and p3LG vector use the internal ribosome entry site from the encephalomyocarditis virus, which allows translation of two consecutive open reading frames from the same messenger RNA.
2. The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. Positive single clone cells were selected with G418 and by PCR. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLTF protein. Analyses of expression efficiencies of selected G418-resistant clones showed that the expression of hLTF mRNA and recombinant protein were higher in CHO and HEK293 cells than those in COS7 cells. But no significant difference between the three cells lines in expression protein. However, the COS7 are suitable for transient expression target recombinant protein. The result of antibacterial experiment indicates that eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA transfection into the mammalian cells CHO, HEK293 cells and COS7 cells and these cells can expression human lactoferrin in high efficiency, which provides the basis for further research of recombinant human lactoferrin protein in mammalian. The recombinant hLTF protein has the effect of inhibiting E. coli proliferation; moreover, its activity is superior to the commercial available hLTF standard proteins.
3. To obtain the recombinant adenoviruses, we developed a system that simplifies the generation and production of adenoviruses containing human lactoferrin transgene in bacteria. The hLTF cDNA was obtained from above steps and was sub cloned into a shuttle vector. After sequence confirmation, the resultant plasmid was linearized by the restriction endonulease Pme co and I transformed with the super coiled adenoviral vector pAdeasy into Escherichia coli strain BJ5183. Recombinants were confirmed by colony PCR and restriction endonulease digestion. The adenoviral vector Ad-LTF was propagated in HEK 293 cells and purified by Cesium chloride density centrifugation. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.
4. The Cre-lox recombination system ahs the potential to become a powerful tool the conditional and cell-specific deletion of genes. The introduction of this system into produces the recombinant adenovirus. First, the hLTF cDNA fragment was cloned into plasmid pDC311 vector, and after sequence confirmation, the resultant plasmid and the plasmid pBHGlox (delta) E1, 3Cre co-transfection on HEK 293 cells. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using Cre-lox system results in highly efficient viral production in HEK 293 cells. Ad-LTF can be further used in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.
5. The recombinant adenovirus containing human lactoferrin cDNA was amplified in HEK293 for four times. Then the final title of recombinant adenovirus was purified. A direct instillation of a recombinant adenoviral vector containing an expression cassette for the human lactoferrin into the mammary gland of rabbits and goats allowed for the efficient secretion of human lactoferrin in the milk. Western blot analysis were performed to confirm hLTF expression after the adenovirus infection on 48h up to 10 d. Through this approach we were able to express human lactoferrin high levels in the milk of rabbits and goat milk and the recombinant protein expression up to 7-10d. This technology platform still to be improved to performed in transgene protein production in the milk of animals.
Conclusions: (1) Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue and four mammalian expression vectors were constructed successfully. (2) The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. These cells can expression human lactoferrin in high efficiency. (3) We developed a method of produce the Ad-hLTF recombinant adenoviruses by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest. (4) We established a method for produce the Ad-hLTF recombinant adenoviruses by using Cre-lox system in mammalian cells. These modifications result in highly efficient viral production systems, which can be further used to produce any recombinant adenovirus containing target gene. (5) We obtained high levels expression functional recombinant hLTF protein in the milk of rabbits and goats.
针对上述技术体系缺陷,本研究探索了以腺病毒为载体,直接导入动物乳腺表达生产转基因蛋白药物的方法。目标基因在动物乳腺上皮细胞中表达,并分泌到乳汁中,从而在动物乳汁中获得重组蛋白药物。其优势在于:(1)不需要构建乳腺特异表达载体,从而简化了转基因蛋白药物生产程序;(2)以腺病毒为载体直接导入乳腺,可获得高表达的目的蛋白药物;(3)乳腺组织可以对人体蛋白质进行正确的修饰和转录后加工,蛋白药物产品的生物学活性接近天然产品;(4)目标蛋白药物在乳腺中高水平短期表达,无需对动物进行长期维持培育,可降低生产成本。
腺病毒载体以其高效转移并表达目标基因和安全性高诸多优点被应用于基因转移之中。但是,国内外有关腺病毒载体的应用一直侧重于基因治疗方面。目前国际上有关腺病毒基因治疗的临床实例应用已上千种,而国内近3年来也已批准了3个有关腺病毒载体应用于人类基因治疗的基因工程药物。但是,迄今为止,腺病毒载体在重组药物蛋白生产方面至今未见到相关应用报道。为此,本研究以人乳铁蛋白重组腺病毒为载体,进行了乳腺表达生产人乳铁蛋白的试验研究,以期探索出高效、简便的转基因蛋白药物生产方法,从而为转基因蛋白药物生产提供新的途径。
本研究共分为五个部分,第一、二部分进行了人乳铁蛋白基因表达载体的构建及人乳铁蛋白cDNA的体外细胞表达,为后面目标基因在动物体内的表达奠定了基础。第三、四部分主要对2种腺病毒载体的构建方法进行了探讨,获得了人乳铁蛋白重组腺病毒高效制备的方法,为进一步利用腺病毒为载体进行乳腺表达奠定了基础。第五部分利用构建成功的重组人乳铁蛋白腺病毒载体,乳腺表达生产人乳铁蛋白蛋白,探讨腺病毒为载体生产转基因蛋白药物的可行性、稳定性及高效性。
第一部分人乳铁蛋白cDNA序列的克隆及真核表达载体的构建实验中,首先从人乳腺组织分离提取得到总RNA,利用RT-PCR方法,直接获得人乳铁蛋白的全长cDNA序列,经DNA测序分析,所得hLTF cDNA与GeneBank中一致。并对克隆的人乳铁蛋白基因的编码氨基酸序列进行分析,结果表明,所得人乳铁蛋白基因序列编码的氨基酸与已发表的人乳铁蛋白编码序列完全一致。以pcDNA3.1、pIRES、pDC316为基础质粒,构建了含有人乳铁蛋白基因的重组4种真核表达载体p3LF、pDCLF、pLG和p3LG。其中,真核表达载体p3LF和pDCLF,含有新霉素抗性筛选标记,可进行转染哺乳动物细胞的抗性筛选,真核表达载体pLG和p3LG,内含有GPF报告基因,可对目标蛋白基因的表达进行实时观测,极大提高了目标蛋白基因检测效率,为后面人乳铁蛋白的表达研究奠定了基础。第二部分人乳铁蛋白cDNA的细胞表达实验中,将上述构建的人乳铁蛋白真核表达载体p3LF、pDCLF、pLG和p3LG分别在COS-7、CHO、和HEK293细胞3种不同细胞株中进行了蛋白表达实验。比较不同真核表达载体在不同哺乳细胞株中的表达效率方法。将其转染进细胞后,CHO和HEK293细胞经G418抗性筛选,获得阳性克隆细胞,并克隆化培养,RT-PCR和Western blotting分析显示,培养上清液中分别有较高的hLTF mRNA和蛋白表达,而COS-7细胞未经G418筛选,直接于转染后48h开始检测细胞培养上清,也获得了蛋白表达。体外抑菌实验证实,重组人乳铁蛋白具有较强的抗菌活性。初步建立了体外真核细胞蛋白表达体系,为后面人乳铁蛋白腺病毒载体构建研究奠定了基础。第三部分细菌内高效制备重组人乳铁蛋白腺病毒Ad-hLTF载体的研究,将体外真核表达验证正确的人乳铁蛋白基因亚克隆至穿梭质粒pShuttle-cmv中,酶切鉴定后所得重组质粒pShuttle-hLTF,经PmeI酶切线性化后,与腺病毒骨架质粒pAdeasy共转化大肠杆菌BJ5183,所获阳性菌落酶切鉴定正确后,获得的重组人乳铁蛋白腺病毒载体Ad-hLTF,经PacI消化后,将大片段纯化后转染HEK293细胞,包装获得重组人乳铁蛋白腺病毒。PCR鉴定病毒上清含有目标蛋白基因hLTF,Western blotting检测到感染细胞上清中含有人乳铁蛋白的表达。结果表明,利用细菌内同源重组法制备的Ad-hLTF重组腺病毒载体是一种方便、高效的方法,所制备的重组腺病毒在体外能有效表达具有生物活性的hLTF,为后面体内实验研究奠定了基础。
第四部分细胞内高效制备重组人乳铁蛋白腺病毒Ad-hLTF载体的研究,为探索利用真核细胞内同源重组生产腺病毒方法,该方法基本原理是通过Cre-loxP或FLP-frt重组酶,使转染进293细胞的Shuttle质粒和辅助大质粒发生定点重组,产生重组腺病毒。得到的重组病毒是E1/E3缺失的复制缺型腺病毒。将目标蛋白基因hLTF克隆至质粒pDC315,该质粒MCS两侧包含有Cre-loxP位点,获得的重组质粒pDC311-hLTF,将其与辅助腺病毒质粒pBHGlox(delta)E1,3Cre共转染HEK293细胞,获得真核细胞内重组包装重组腺病毒Ad-hLTF,PCR鉴定病毒上清含有目标蛋白基因hLTF,Western blotting检测到感染细胞上清中含有人乳铁蛋白的表达,体外抑菌实验结果表明,重组人乳铁蛋白具有较强的抗菌活性。结果表明,利用真核细胞内同源重组法制备的Ad-hLTF重组腺病毒载体是一种更为简便的方法,省去了细菌重组构建大质粒的筛选鉴定过程,所制备的重组腺病毒在体外能有效表达具有生物活性的hLTF。
第五部分重组腺病毒Ad-hLTF在兔和羊乳腺中高效表达的研究,将上述构建正确的重组hLTF腺病毒载体,在HEK293细胞中经过4轮扩增,所获病毒经进一步纯化后,最终获得病毒滴度1011-1012PFU/ml。通过将重组hLTF腺病毒在兔和羊的乳腺中的体内表达实验,48 h后分别检测到了兔和羊奶中的hLTF的高表达。结果表明,所构建的重组hLTF腺病毒载体在兔和羊乳腺中均获得了高效表达,Western blotting检测病毒感染后2-10 d的兔和羊的乳汁,可检测到人乳铁蛋白的表达,从Western blotting人乳铁蛋白的特异条带结果分析,兔乳汁中人乳铁蛋白的表达量高于羊乳汁中hLTF的表达量。该体系尚待进一步改进和不断完善。下一步将继续探索腺病毒为载体在羊乳腺中的高效表达。以此,建立腺病毒为载体,乳腺表达生产转基因蛋白药物的技术体系。结论:(1)应用RT-PCR方法直接从人乳腺组织中扩增获得人乳铁蛋白cDNA全序列;(2)构建了4种不同的人乳铁蛋白真核表达载体;(3)获得了人乳铁蛋白cDNA在细胞的体外表达;(4)建立了细菌内同源重组高效制备人乳铁蛋白腺病毒载体的方法,并获得了表达有生物活性的人乳铁蛋白腺病毒载体;(5)建立了细胞内同源重组高效制备人乳铁蛋白腺病毒载体的方法,并获得了表达有生物活性的人乳铁蛋白腺病毒载体;(6)获得了重组人乳铁蛋白腺病毒在兔和羊乳腺中的高效表达。
本研究首次以腺病毒为载体,在动物乳腺中,获得了重组人乳铁蛋白的高效表达,为重组人乳铁蛋白的生产奠定了基础。该方法进一步拓展了腺病毒载体的应用,是乳腺生物反应器原理生产转基因蛋白药物产品的新应用。该方法无论在转基因技术理论领域,还是在基因工程药物生产方面,均具有其创造性和实用性。
The mammary gland has been identified as a suitable alternative to produce biopharmaceutical and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk from which the purification process is relatively simple. So, transgenesis has been extensively used for the genetic modification of farm animals to produce human recombinant proteins in milk. This system concluding four steps:(i)First,construct the recombinant expression vectors and special sequences are specific for mammary glad expression; But it is known that this control system is not absolute, allowing for transgene expression founders might die even in the fetal stage, where only the animals in which the expression levels were very low were born. (ii) Through some transgene methods and get the recombinant embryo or cells. (iii) Embryo transfer and nuclear transfer and got the transgene animal ;(vi) after breeding the transgene animals, which to be detected for expression target gene.
However, drawbacks in this technology include (i) the technically difficult procedure required, its low efficiency in produce transgenic animals;(ii) especially its inefficiency when applied to large livestock, (iii)the length of time between incorporating foreign DNA and harvesting the exogenous protein,(vi)and the health risk for animals due to the ectopic expression of transgenes. The direct in vivo transfection of the mammary gland has been proposed as a faster and more inexpensive alternative to target the expression of a heterologous gene to the secretory mammary epithelial cells. Although these expression levels may be enough for certain basic studies, they must be improved when large amounts of recombinant proteins are required.
Build on above traditionally technology system shortcomings,this paper reported a strategy to produce transgenic recombinant protein in the milk of animal mammary gland, such as rabbits and goats. Adenoviral vectors are able to successfully infect the mammary glandular epithelium and to promote the secretion of high amounts of a recombinant protein in the milk of animals. This system has some merits (i) the direct transduction of the mammary gland with adenoviral vectors could be a very useful tool to study the powerfulness of mammary-specific promoters avoiding variables associated to the position effects or to the copy numbers. The use of constitutive promoters, undoubtedly simplifies vector design, however this does not preclude the use of tissue specific promoters for special applications. (ii) It is possible to obtain high expression levels of a foreign protein in milk. (iii) The mammary gland can produce an in vivo biologically active recombinant protein. (vi) The secretion of the recombinant human lactoferrin in the milk is not permanence expression, so have low products cost.
Members of the adenovirus family infect a great variety of post-mitotic cells; even those associated with highly differentiated tissues such as skeletal muscle, lung, brain and heart. Since they deliver their genome to the nucleus and can replicate with high efficiency, they are prime candidates for the expression and delivery of therapeutic genes. They have a wide host-range and are currently divided into three genera with further subdivision into species (also termed subgenera or subgroups) A to F. Division of human stereotypes, based mainly on immunological criteria, has historically been the basis of classification
Recombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy. Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. The ability of adenovirus vectors to deliver and express genes at high yields, especially in vitro, however, to date, there has little reports have been used adenovirus for production of transgenic recombinant protein. The objective of our study is to investigate a new method by using recombinant adenovirus, which can produce the biopharmaceutical, and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk. The method may be used also to assess the functionality of gene constructs prior to the generation of a transgenic animal, and may be a viable approach to study the physical, chemical and biological characteristics of recombinant proteins expressed in milk of different species at the same time.
In order to establish a platform for the development of new biotechnological processes directed to the production of recombinants complex proteins in milk. We have carried out the studies on the cloning of objective gene and construction of mammalian expression vector and recombinant adenovirus containing hLTF cDNA, also, the rAd vectors direct transduction of the mammary gland. The main results are as following:
1. Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue, and then subcloned into pcDNA3.1 vector. hLTF cDNA sequence was determined, which consists of 2169bp. Comparison with five other hLTF cDNA sequences registered in GeneBank shows 99 % homology in DNA sequence. Four mammalian expression vectors were constructed, the pLG and p3LG vectors have GFP report gene and the incorporation of a GFP protein in this vector was designed to serve as a live marker for tracking infected cells or tissues in animal studies. The vectors p3LF and pDCLF use the neomycin resistance gene (Neor) to permit selection of transformed cells. The pLG plasmid and p3LG vector use the internal ribosome entry site from the encephalomyocarditis virus, which allows translation of two consecutive open reading frames from the same messenger RNA.
2. The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. Positive single clone cells were selected with G418 and by PCR. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLTF protein. Analyses of expression efficiencies of selected G418-resistant clones showed that the expression of hLTF mRNA and recombinant protein were higher in CHO and HEK293 cells than those in COS7 cells. But no significant difference between the three cells lines in expression protein. However, the COS7 are suitable for transient expression target recombinant protein. The result of antibacterial experiment indicates that eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA transfection into the mammalian cells CHO, HEK293 cells and COS7 cells and these cells can expression human lactoferrin in high efficiency, which provides the basis for further research of recombinant human lactoferrin protein in mammalian. The recombinant hLTF protein has the effect of inhibiting E. coli proliferation; moreover, its activity is superior to the commercial available hLTF standard proteins.
3. To obtain the recombinant adenoviruses, we developed a system that simplifies the generation and production of adenoviruses containing human lactoferrin transgene in bacteria. The hLTF cDNA was obtained from above steps and was sub cloned into a shuttle vector. After sequence confirmation, the resultant plasmid was linearized by the restriction endonulease Pme co and I transformed with the super coiled adenoviral vector pAdeasy into Escherichia coli strain BJ5183. Recombinants were confirmed by colony PCR and restriction endonulease digestion. The adenoviral vector Ad-LTF was propagated in HEK 293 cells and purified by Cesium chloride density centrifugation. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.
4. The Cre-lox recombination system ahs the potential to become a powerful tool the conditional and cell-specific deletion of genes. The introduction of this system into produces the recombinant adenovirus. First, the hLTF cDNA fragment was cloned into plasmid pDC311 vector, and after sequence confirmation, the resultant plasmid and the plasmid pBHGlox (delta) E1, 3Cre co-transfection on HEK 293 cells. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using Cre-lox system results in highly efficient viral production in HEK 293 cells. Ad-LTF can be further used in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.
5. The recombinant adenovirus containing human lactoferrin cDNA was amplified in HEK293 for four times. Then the final title of recombinant adenovirus was purified. A direct instillation of a recombinant adenoviral vector containing an expression cassette for the human lactoferrin into the mammary gland of rabbits and goats allowed for the efficient secretion of human lactoferrin in the milk. Western blot analysis were performed to confirm hLTF expression after the adenovirus infection on 48h up to 10 d. Through this approach we were able to express human lactoferrin high levels in the milk of rabbits and goat milk and the recombinant protein expression up to 7-10d. This technology platform still to be improved to performed in transgene protein production in the milk of animals.
Conclusions: (1) Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue and four mammalian expression vectors were constructed successfully. (2) The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. These cells can expression human lactoferrin in high efficiency. (3) We developed a method of produce the Ad-hLTF recombinant adenoviruses by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest. (4) We established a method for produce the Ad-hLTF recombinant adenoviruses by using Cre-lox system in mammalian cells. These modifications result in highly efficient viral production systems, which can be further used to produce any recombinant adenovirus containing target gene. (5) We obtained high levels expression functional recombinant hLTF protein in the milk of rabbits and goats.
引文
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