【作者】 成勇；

【导师】 谢庄；

【作者基本信息】 南京农业大学 ， 遗传学， 2007， 博士

【摘要】 由于高质量生物药品和医学诊断的需求,转基因动物乳腺生物反应器的研究已经成为生物工程领域研究的热点之一;成为高效、高质量生产昂贵生物活性蛋白的有效途径之一。应用乳腺生物反应器生产人的重组蛋白较其他的生物学制备系统有产品活性高、成本低的优点,因而某些人的重组蛋白更适宜用乳腺生物反应器来生产。外源基因在转基因动物乳腺中特异性表达是转基因动物乳腺生物反应器的根本目的。有许多生物活性蛋白基因在乳蛋白调控序列的指导下已经在转基因动物乳腺中表达,其中应用较多的调控元件有:牛αs1-酪蛋白（bovineαs1-casein）、山羊β-酪蛋白（β-casein）、绵羊β-乳球蛋白（BLG）、小鼠的乳清酸蛋白（WAP）等。尽管已有应用这些调控序列实现乳腺特异性表达成功的事例,但大多数实验研究中,乳腺特异性表达的成功率低、乳汁中的表达水平低。已经发现某些非乳蛋白调控元件对乳腺特异性表达有明显的作用,如绝缘子、染色质开启子、增强子、核基质附着区和内含子等,其中起到重要作用的是转录和翻译调控因子。近年来,尽管乳蛋白启动子及其它表达调控元件已广泛应用于乳腺特异性表达,但相对低的表达水平和不稳定表达案例也较多。人巨细胞病毒（CMV）启动/增强子是广泛用于真核细胞表达的调控元件,早期的研究证明有提高转录水平的作用,但未见报道用于激活乳腺特异性表达。本研究应用人巨细胞病毒启动/增强子（Pcmv）与乳蛋白调控元件构建复合启动/增强子和人乳铁蛋白cDNA组成乳腺特异性表达载体。为了鉴定复合启动/增强子对转基因动物乳腺特异性表达水平的作用,我们构建了单一乳蛋白启动子与乳蛋白/Pcmv复合启动子两种不同类型的乳腺特异性表达载体。应用山羊β-酪蛋白、山羊β-乳球蛋白、αs1-牛酪蛋白为乳蛋白调控序列为乳蛋白调控序列;鸡β-珠蛋白（β-globin,隔离元件）、CMV启动/增强子和SV40polyA为非乳蛋白启动/增强子。以不同的乳蛋白调控序列与启动/增强子组合构建乳腺特异性表达复合启动/增强子（chimeric promoter/enhancer）,单一的酪蛋白启动子作为对照,与人乳铁蛋白cDNA组成乳腺特异性表达载体。共构建了7个乳腺特异性表达载体（BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36）,其中4个为复合启动/增强子型载体,另3个为单一乳蛋白启动子型载体。BnF95、pBnCL14和pBnLC2G载体用于转染山羊乳腺上皮细胞（goat mammary epithetical cells,GMECs）;pgCNCG/LF25和pbCNCS/LF36为复合启动/增强子型,其中pgCN/LF/g和pbCN/LF为单一酪蛋白启动子作为对照,用于制备转基因小鼠。泌乳山羊乳腺组织经Ⅰ型胶原酶消化分离乳腺上皮细胞,DMEM/F₁₂培养液（10%FCS）传代培养,获得的乳腺上皮细胞最多可培养25代以上。采用分步胰酶消化法去除上皮细胞中所含的成纤维细胞,从而乳腺上皮细胞得到纯化。通过电转染法将含NEO^τ标记基因的BnF95、pBnCL14和pBnLC2G外源基因导入23个山羊乳腺上皮细胞系,经G418筛选三周后,得到有抗性的转染细胞株,挑取单克隆细胞株进行增殖培养。共获得242株转染外源基因的细胞,有118株转染pBnCL14,82株转染BnF95,42株转染pBnLC2G基因构件。有3株转染pBnCL14基因的细胞进行nestedPCR整合检测,结果均为外源基因整合阳性。初步认为经G418抗性培养后获得细胞为整合细胞。对抗性培养阳性的细胞株应用催乳素进行诱导表达,收集48和72小时的诱导细胞培养液进行ELISA检测,其中有30株细胞的诱导液中检测出重组人乳铁蛋白,有4株转染pBnCL14基因细胞的表达水平达到10-50mg·L^-1;另有22株细胞（9株/BnF95,7株/pBnLC2G）表达水平低于10 mg·L^-1。通过G418筛选和诱导表达和ELISA检测验证具有表达功能的乳腺上皮将用于山羊体细胞克隆的供核细胞。pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36四个构件应用胚胎显微注射的方法制备转基因小鼠。应用PCR初步筛选整合小鼠,获得14只原代转基因小鼠,PCR产物序列分析显示转基因小鼠PCR检测产物与模板的同源率为99.95%～99.75,在PCR正常正确率范围以上。原代鼠与C57或ICR正常鼠交配,在出生的后代鼠中检出37只F1代转基因小鼠。获得的转基因母鼠用ELISA检测乳汁中重组人乳铁蛋白,并繁殖传代,获得F1、F2代转基因小鼠。复合启动/增强子构件指导人乳铁蛋白cDNA在原代转基因小鼠乳腺中重组人乳铁蛋白（rhLF）表达水平(2～8.1g·L^-1)明显高于单一酪蛋白启动子构件(0～12mg·L^-1),约10000倍。在所有转基因小鼠的血清和唾液中未检测（ELISA）出重组人乳铁蛋白（rhLF）。研究表明,酪蛋白/Pcmv复合启动/增强子不仅可提高转基因小鼠乳腺特异性表达水平,而且具有良好的乳腺组织表达定位性。近年来,对基因表达调控机制提出了一些新的看法,认为几乎所有的哺乳动物基因表达是由多个环节调控的,其中包括了转录、转录后加工、核输出和定位、mRNA分子的成熟、翻译和稳定性等。生物信息学分析显示Pcmv含有多个uORF和CCAAT增强子结合位点。Lodhi等（2003）认为uORF能介导编码mRNA翻译的作用,而CCAAT序列是增强子结合蛋白结合的位点。有研究报道,CCAAT位点结合增强子结合蛋白也可介导功能基因的表达,这一过程可能意味着在5’-UTR顺式调控元件,包括复合启动子中乳蛋白和CMV启动子序列能激活人乳铁蛋白cDNA在培养的山羊乳腺上皮细胞和转基因小鼠乳腺中表达。本领域的研究报道显示,尽管已有一些利用长片段调控元件获得高表达的事例,但更多的低表达案例使开发那些构建方便、片段长度短小、表达效率和表达水平高的乳腺特异性表达载体显得十分迫切。本研究的结果不仅实现了cDNA功能基因的高水平、高效乳腺特异性表达,而且其载体的构建简单方便。到目前为止,尚未见有应用乳蛋白/Pcmv复合启动子激活cDNA功能基因实现乳腺特异性高效表达的报道。更多还原

【Abstract】 The use of transgenic animals for mammary bioreactors is a rapidly developing and intensely interesting field due to the increased demand for high-quality pharmaceuticals and medically diagnostic preparations.The manufacture of recombinant human proteins is more profitable by mammary gland bioreactor than that by other biological systems.The mammary gland-specific expression of a transgene is a major objective for producing heterogenous proteins in the milk of transgenic animals.Bio-active foreign proteins have been expressed specifically in the mammary under the direction of particular milk regulatory sequences,such as:goatβ-casein,bovineαs1-casein,ovineβ-lactoglobulin （BLG） and rat whey acid protein（WAP）.However,much of the evidences regarding the mammary as bioreactor indicated that only a small percentage of the mammary glands of transgenic animals expressed functional protein,and most of them were found at low levels in milk.Several major regulatory elements are required in order to obtain satisfactory transgene expression,such as essential gene insulators,chromatin openers,enhancers,matrix attached regions,and introns.One of the most important elements may be the factor controlling the transgene’s transcription and translation.In recent years,although lactoprotein gene promoters were widely used,it should be noted that their expression levels in milk were relatively low and unstable in many instances.As an alternative to the widely used cytomegalovirus（Pcmv） enhancer for expressing transgene in eukaryon,there was no report for promoting transgenic expression in mammary gland,our aim was to build a series of promoters bearing at least one,or if possible,all of the following characteristics:promoters that would increase high levels of expressing transgene in milk,and expression specifically located in the mammary organs. We used the chimeric promoters/enhancers combining sequences of lactoprotein promoters from cow and goat,with the Pcmv or mono-milk-promoter without Pcmv or SV40ployA to construct vectors for expressing human lactoferrin（hLF） cDNA,their ability to drive high levels of specific recombinant human lactoferrin（rhLF） expression was determined in transgenic mice or goat mammary epithelial cells（GMECs） in vitro.Vectors of mono-promoters of lactoprotein elements were constructed as a control.Seven vectors （BnF95,pBnCL14,pBnLC2G,pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36） with different promoters/enhancers were constructed.Three vectors of BnF95,pBnCL14 and pBnLC2G were constructed by using milk regulatory elements of goat BLG and neomycin resistance marker gene（NEO^r） for transfecting of GMECs;pgCNCG/LF25,and pbCNCS/LF36 were constructed by using promoter enhance sequences of goat casein, bovineαs1-casein,chickenβ-globin insulators,Pcmv and SV40polyA for producing transgenic mice.pgCN/LF/g and pbCN/LF contained mono-promoters of casein as controls.Primary GMEC lines were derived from biopsy tissue from artificial lactating goats. The cells were cultured in DMEM/F₁₂ containing 10%fetal calf serum（FCS）.GMECs could keep proliferate vigorously even 25 passages in vitro.The pure GMECs lines could be obtained within 2-3 passages by multi-alternation of digesting and adhering repeatedly. By electroporation,BnF95,pBnCL14 and pBnLC2G encoding neomycin resistance for selecting the positive cells were transfected into GMECs in vitro.After three-weeks-culture by G418(400ng·L^-1),the transfected cell colonies grew and proliferated in G418 medium. Single colonies were picked into another 24-well plate for continue culture,and 242 colonies were generated from 118 clonies of GMECs by neo-resistant culture.Three of cell clones from 242 colonies were respectively screened by PCR and each was confirmed to be transgenic colonies corresponded with NEO^r ones,the total 82,42 and 118 colonies were transfected transgenes of pBnCL14,BnF95 and pBnLC2G respectively.These colonies were induced with luteotropic hormone,and then the media were gleaned respectively at 48h or 72h.It was found that there were 30 colonies expressing recombinant human lactoferrin（rhLF） by ELISA at 72h.4 colonies of higher expression level of rhLF were about 50mg·L^-1 from transgene of pBnCL14.These GMECs of expressing rhLF would be used as donor cells for hircine somatic cell nuclear transfer（SCNT） to produce the transgenic goats.Four vectors（pgCN/LF/g,pgCNCG/LF25,pbCN/LF,pbCNCS/LF36） were used to generate transgenic mice.A total of 14 lines were selected out of 160 candidates.To verify transgenic integration,these PCR products were sequenced,and the sequences were analyzed using DNAStar software.Compared with correlative transgenes,the percentage of sequence identity was 100 except 99.75 for pgCNCG/LF25 founder line.Founder mice bearing the transgene were mated to C57 males or female and delivered 110 pups,of which 52 progeny were identified as being transgenic.Using ELISA detection,results showed that the recombinant human lactoferrin rhLF expression was significantly increased in the milk of transgenic mice with chimeric promoter transgenes.The expression level was 2-8.1g·L^-1 in the milk using chimeric promoter.In contrast,only 0～0.012g·L^-1 was found in the milk using mono-casein promoter.In two of the highly expressing lines,pbCNCS/LF36 and pgCNCG/LF25,there was approximately a 10,000-fold increase in target protein in the milk of transgenic mice （P＜0.001）.In addition,we have also shown that the expression of rhLF was strictly limited to the mammary organs in both founders and F1 offspring,because no rhLF was detected in the saliva or blood of transgenic mice expressing high levels rhLF in milk.This result suggested that the constructs with chimeric promoter/enhancer could increase the expression of heterogenous protein in the mammary gland of transgenic animals with high specificity.Recently,continuing discoveries of new and surprising mechanism of gene regulation suggest many and perhaps all genes are regulated at multiple steps including transcription, post-transcriptional processing,nuclear export and localization,stability,and translation of mature mRNA molecules.Chimeric promoter/enhancer contains both mammary gland specific elements and Pcmv for mammary expression.Bio-informatics analysis shows that Pcmv sequence contains 10 uORF（upstream open reading frame） and 4 CCAAT/enhancer sites.Lodhi et al（2003） considered that the uORF could mediate constitutive effects on translation of mRNA from AUG codon.CCAAT sequence is an enhancer-binding site having been described in being linked to CCAAT-binding protein,some research show the increasing of the binding would mediate expression of functional gene,which might underlie observation that some cis-acting elements within the 5’-UTR including chimeric promoters of lactoprotein and CMV can activate initiation of translation.From overall results,pgCNCG/LF25（composed of Pcmv chickenβ-globin and goatβ-casein promoter）,pbCNCS/LF36（Pcmv,SV40polyA and bovineαs1-casein promoter）,pBnCL14（Pcmv goatβ-BLG promoter AND SV40polyA） and pBnLC2G （Pcmv,2×chickenβ-globin and goatβ-BLG） were the most efficient under in vivo and in vitro conditions.The pBnCL14 was the most efficient in BnF95 in the expression of GMECs in three of pBnCL14,pBnCL14 and pBnLC2G,while the PgCNCG/LF25 was more efficient than the pbCNCS/LF36 in mammary gland of transgenic mice.It is worth mentioning that the binary promoter of Pcmv can improve mammary gland-specific expression,while ternary promoter of CMV,chickenβ-globin and lactoprotein may be more active than the binary one in mammary gland of transgenic mice.Conclusively,although many long regulatory sequence can be useful for driving expression of targeted transgene in milk,their low efficiencies and uncertain expression,in most transgenic individuals,caused researchers to concentrate much time and effort on constructing over-expressing vectors for mammary specific-gland expression.Compared to constructing large size regulatory elements,the chimerc promoters/enhancers for conveniently constructing mammary-specific expression vectors,were shown to ascribe a high level of stability and efficiency of expression.We describe for the first time the synergistic effect achieved by the use of binary or ternary promoters/enhancers for mammary specific expression of rhLF in transfected GMECs and the milk of transgenic mice.更多还原