CN111826389A - 提高大肠杆菌重组蛋白胞外分泌水平的方法 - Google Patents
提高大肠杆菌重组蛋白胞外分泌水平的方法 Download PDFInfo
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Abstract
本发明涉及一种提高大肠杆菌重组蛋白胞外分泌水平的方法,通过同源重组的方法将包含1‑4个SD序列的基因片段整合至编码D‑丙氨酰‑D‑丙氨酸羧肽酶DacA的启动子与DacA编码基因之间。本发明通过整合不同的SD序列至大肠杆菌DacA基因的启动子与DacA编码基因之间,提高了D,D‑羧肽酶DacA的表达,从而提高大肠杆菌重组蛋白胞外分泌水平。
Description
技术领域
本发明涉及基因工程与发酵工程领域,尤其涉及一种提高大肠杆菌重组蛋白胞外分泌水平的方法及重组大肠杆菌。
背景技术
大肠杆菌表达***是目前基因工程中最常用的重组蛋白表达***,有操作简单、成本低廉等优点,可以快速大规模地生产目的蛋白,但是在大肠杆菌的异源表达中,大部分重组蛋白在信号肽的引导下分泌到周质空间,会导致中间体大量积聚,对蛋白质的生产造成阻碍。
肽聚糖是由双糖单位,四肽尾还有肽桥聚合而成得多层网状大分子结构。肽聚糖层的骨架由N-乙酰葡萄糖胺和N-乙酰胞壁酸通过β-1,4糖苷键连接而成,糖链间由肽链交联,构成稳定的网状结构,这样构成了整个细菌表面的细胞壁。作为细胞壁的主要成分,肽聚糖的组成与细胞结构的完整性、外膜的通透性的维持、细菌的抗干燥能力和耐热性等均密切相关。
CN105755029A公开了一种基于dacA提高大肠杆菌重组蛋白胞外分泌水平的方法,通过过表达dacA基因,获得重组蛋白胞外分泌水平过表达的重组大肠杆菌。
启动子是转录起始的位点,是RNA聚合酶的结合位点,在基因的表达过程中起着重要的调节作用。启动子与RNA聚合酶的结合强度以及其与各种转录因子的相互作用直接影响下游基因的转录水平以及表达水平。1974年Shine和Dalgarno首先发现,在mRNA上有核糖体的结合位点,它们是起始密码子AUG和一段位于AUG上游3~10bp处的由3~9bp组成的序列。这段序列富含嘌呤核苷酸,刚好与16S rRNA 3’末端的富含嘧啶的序列互补,是核糖体RNA的识别与结合位点。以后将此序列命名为Shine-Dalgarno序列,简称SD序列。它与起始密码子AUG之间的距离是影响mRNA转录、翻译成蛋白的重要因素之一,某些蛋白质与SD序列结合也会影响mRNA与核糖体的结合,从而影响蛋白质的翻译。因此对启动子进行改造是提高大肠杆菌重组蛋白胞外分泌的重要方法。
CN 104031913A公开了一种用于在枯草芽孢杆菌中分泌表达外源蛋白的表达设备,包括以下P43启动子、SD序列、信号肽、多克隆位点和蛋白纯化标签,该表达设备可实现异源蛋白在枯草芽孢杆菌中的高效分泌表达。CN 106591309A公开了一种茶碱诱导型基因表达***,以组成型强启动子P43为基础组件,融合经过改造了人工SD元件和SD序列和起始密码之间的间隔区长度的茶碱控制元件riboE1,构建出茶碱诱导型基因表达元件P43-riboE1,并进一步改造后使外源基因在茶碱控制下实现高水平表达。CN102533833A公开了一种链霉素表达质粒的构建方法及角蛋白酶的生产方法,其构建的表达质粒在变铅青链霉菌中表达后角蛋白酶产量高于出发菌株。
但是,对不同的菌株SD序列的位置及其与起始密码子AUG之间的距离具体应当如何设置,由此对重组蛋白胞外分泌的影响并不十分明确。因此,开发越来越多的启动子改造方法以提高大肠杆菌重组蛋白胞外分泌水平十分必要。
发明内容
为解决上述技术问题,本发明的目的是提供一种提高大肠杆菌重组蛋白胞外分泌水平的方法,本发明通过整合不同的SD序列至大肠杆菌DacA基因的启动子上,提高了D,D-羧肽酶DacA的表达,从而提高大肠杆菌重组蛋白胞外分泌水平。
本发明的第一个目的是提供一种提高大肠杆菌重组蛋白胞外分泌水平的方法,通过同源重组的方法将包含1-4个SD序列(优选为1-2个SD序列)的基因片段整合至编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子与DacA编码基因之间。
进一步地,编码所述SD序列的核苷酸序列如SEQ ID No.1所示。
进一步地,距离DacA初始密码子ATG最近的SD序列与DacA的初始密码子ATG的距离为0-20bp。当距离DacA起始密码子最近的SD序列与初始密码子ATG序列的距离为0bp时,即表示距离DacA初始密码子ATG最近的SD序列与DacA初始密码子ATG之间没有任何碱基序列。
进一步地,大肠杆菌是E.coli BL21、E.coliJM109、E.coli DH5α、E.coliRosetta、E.coli TOP10、E.coliM110、E.coliS110等。优选地,大肠杆菌是E.coli BL21。
进一步地,整合方法包括以下步骤:
(1)设计并合成含有卡那抗性基因及待整合基因上下游同源臂的整合框;所述待整合基因包括编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子,所述上下游同源臂中包括1-4个SD序列;
(2)将整合框电转化至含有质粒载体的大肠杆菌电转化感受态细胞,同源重组后筛选出基因整合的大肠杆菌,并消除卡那抗性(kan)基因。
进一步地,在步骤(2)中,质粒载体为pKD46质粒。
进一步地,在步骤(2)中,采用pCP20辅助质粒消除kan基因。
优选地,整合方法包括以下步骤:
(1)引入不同数目SD序列时,设计并合成与目的基因有同源片段的引物,PCR扩增获得含有抗性基因并在其两侧含有FRT位点的片段,作为SD序列整合框片段;其中,目的基因包括1-4个SD序列;
(2)将质粒载体转化至大肠杆菌感受态细胞,制备电转化感受态细胞,将电转化感受态细胞与步骤(1)制得的整合框片段混合后进行电转化,同源重组后筛选出含有抗性基因的阳性转化子;
(3)利用pCP20质粒消除抗性基因,得到重组大肠杆菌;与出发菌株相比,该重组大肠杆菌用于生产重组蛋白时,重组蛋白的胞外分泌水平提高。
本发明通过整合不同数目的SD序列到D-丙氨酰-D-丙氨酸羧肽酶的基因dacA的启动子与DacA编码基因之间,提高了重组大肠杆菌外膜的渗透性,提高了D,D-羧肽酶DacA的表达,增加了胞内可溶性肽聚糖,因此提高了重组蛋白胞外分泌水平。
本发明中,SD序列的数目并非越多越好,因为引入的SD序列过多,会导致DacA启动子与DacA编码基因之间的距离增大,过大的启动子与目的基因之间的距离会降低启动子的转录水平及目标基因DacA的翻译水平。
距离DacA的启动子最近的SD序列与DacA的启动子中AUG序列的距离应保持合适范围,优选为0-20bp,更优选为0-4bp,最优选为0bp。
本发明的第二个目的是提供一种重组大肠杆菌,重组大肠杆菌中编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子和DacA编码基因之间整合有1-4个(优选为1-2个)SD序列。
进一步地,编码SD序列的核苷酸序列如SEQ ID No.1所示。
进一步地,距离DacA起始密码子最近的SD序列与起始密码子AUG序列的距离为0-20bp。
进一步地,重组大肠杆菌的出发菌株为E.coli BL21、E.coliJM109、E.coli DH5α、E.coli Rosetta、E.coli TOP10、E.coliM110、E.coliS110等。
本发明的第三个目的是公开上述重组大肠杆菌在重组蛋白胞外生产中的应用。
进一步地,重组蛋白包括淀粉酶(AMY)、绿色荧光蛋(GFP)、胶原蛋白、果糖基转移酶、甲基对硫磷水解酶、过氧化氢酶、葡萄糖氧化酶等。优选地,重组蛋白包括淀粉酶(AMY)或绿色荧光蛋(GFP)。
进一步地,重组蛋白的生产方法为本领域常用生产方法。
借由上述方案,本发明至少具有以下优点:
dacA基因表达的D,D-羧肽酶能够从肽聚糖五肽上移除最后一个末端D-Ala,导致转肽反应中供体不可用,可以控制肽聚糖的网状交联水平,对细胞壁肽聚糖层的合成与结构稳定具有调控作用,从而改变细胞的通透性。本发明通过整合不同数目的SD序列到D-丙氨酰-D-丙氨酸羧肽酶的基因dacA的启动子和DacA编码基因之间,获得重组蛋白胞外分泌水平显著提高的重组大肠杆菌。
本发明提供的重组菌株的胞外蛋白的分泌水平显著提高,本发明方法对重组蛋白在大肠杆菌中的高效分泌与生产具有重要指导意义。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是整合了1SD、2SD、3SD、4SD序列的菌株的菌落PCR验证图。M:2000bp DNAMarker;1:BL21::1SD菌株;2:BL21::2SD菌株;3:BL21::3SD菌株;4:BL21::4SD菌株。
图2图示了基因整合1SD序列至羧肽酶DacA的ATG不同距离对E.coli胞外生产GFP的影响。1SD:BL21(+1SD)-gfp菌株、1SD-2:BL21(+1SD)-2-gfp菌株、1SD-4:BL21(+1SD)-4-gfp菌株、1SD-6:BL21(+1SD)-6-gfp菌株、1SD-8:BL21(+1SD)-8-gfp菌株、1SD-10:BL21(+1SD)-10-gfp菌株。
图3图示了1SD、2SD序列整合对绿色荧光蛋白胞外分泌表达的影响。BL21:BL21-gfp菌株;1SD:BL21(+1SD)-gfp菌株;2SD:BL21(+3SD)-gfp菌株;3SD:BL21(+3SD)-gfp菌株;4SD:BL21(+4SD)-gfp菌株。
图4图示了1SD、2SD序列整合对淀粉酶胞内外分泌表达的影响。(a)淀粉酶酶活力;(b)SDS-PAGE凝胶电泳图;BL21:BL21-amyk菌株;1SD:BL21(+1SD)-amyk菌株;2SD:BL21(+2SD)-amyk菌株;M:标准蛋白Marker;1:BL21-amyk;2:BL21(+1SD)-amyk;3:BL21(+2SD)-amyk。
图5图示了1SD、2SD序列整合对E.coli细胞膜渗透性的影响。BL21:BL21菌株;1SD:BL21::1SD菌株;2SD:BL21::2SD菌株。
图6图示了1SD、2SD序列整合对α-半乳糖苷酶胞外分布的影响。BL21:BL21菌株;1SD:BL21::1SD菌株;2SD:BL21::2SD菌株。
本发明实施例中所用到的材料和方法:
1)培养基
LB固体培养基:15g/L琼脂,10g/L胰蛋白胨,5g/L酵母提取粉,10g/L NaCl,pH7.0。
LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取粉,10g/L NaCl,pH 7.0。
TB液体培养基:12g/L胰蛋白胨,24g酵母提取粉,甘油4mL,2.31g KH2PO4,12.54gK2HPO4,pH 7.5,定容到1L。
2)培养方法
种子培养:将平板划线长出的单菌落接到LB液体培养基中,培养转速200r/min,在37℃下恒温摇床培养8h。
发酵培养:将重组E.coli在LB培养基中37℃培养8h,然后按1%(v/v)的接种量接种到TB培养基中,37℃培养至OD600=0.8,加入终浓度为1mmol·L-1IPTG,25℃诱导表达。氨苄青霉素和氯霉素工作浓度均为50μg·mL-1,卡那霉素工作浓度为25μg·mL-1。
3)分析方法
生物量测定:测定600nm下的吸光光度值。
淀粉酶活性测定:淀粉酶的一个酶活力单位(U)定义:在pH 9.5和50℃下,每分钟水解淀粉释放1μmol还原糖(葡萄糖)所需的酶量。使用改良的二硝基水杨酸(DNS)法测定可溶性淀粉水解过程中产生的还原糖。
绿色荧光蛋白荧光测定:将菌液在4℃、1×104×g条件下离心10min,将上清与细胞分离,将菌体细胞用10mmol·L-1pH 7.4PBS磷酸盐缓冲液重悬,测定其菌浓OD600nm;使用酶标仪和96孔板测定上清中GFP的荧光强度。10mmol·L-1pH 7.4PBS的荧光值作为空白对照。激发和发射波长分别为488nm和533nm。
将100μL酶液与50μL 10mmol·L-1对硝基酚-α-D-吡喃半乳糖(p-NPG)和50μLpH5.8 100mmol·L-1磷酸氢二钠-柠檬酸缓冲液混匀,反应15min后(45℃下),即刻加入3mL碳酸钠(浓度为0.25mmol·L-1)终止反应。用酶标仪测定400nm处的吸光值。
本发明以下实施例中,编码SD序列的核苷酸序列如SEQ ID No.1所示。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1整合不同数目SD序列的重组大肠杆菌的构建过程
(1)以质粒pKD13为模板,设计引物并PCR扩增含有Kan抗性基因的用于替换1SD、2SD、3SD、4SD序列的同源片段,序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQID NO.4所示,其中,Kan抗性基因两侧含有FRT位点,得到不同的SD序列整合框片段。
(2)将pKD46质粒转化至E.coli BL21(DE3)感受态细胞,获得E.coli BL21(DE3)/pKD46菌株,制备电转化感受态细胞,将步骤(1)制得的整合框片段电转化进入E.coli BL21(DE3)/pKD46感受态中,转化液经过后培养后涂布含有卡那霉素(Kan)、氨苄青霉素的LB固体培养基中培养,获得转化子BL21::1SD::kan、BL21::2SD::kan、BL21::3SD::kan、BL21::4SD::kan。通过菌落PCR扩增验证SD序列是否成功整合,筛选出含有卡那霉素抗性基因的阳性转化子E.coli BL21(DE3)::1SD::kan/pKD46、E.coli BL21(DE3)::2SD::kan/pKD46、E.coli BL21(DE3)::3SD::kan/pKD46、E.coli BL21(DE3)::4SD::kan/pKD46。理论上,BL21::1SD::kan、BL21::2SD::kan、BL21::3SD::kan、BL21::4SD::kan应分别含有1892、1898、1904、1910bp的同源片段,如图1所示,菌落PCR获得的片段大小与理论上同源片段的大小相符。
(3)将E.coli BL21(DE3)::1SD::kan/pKD46、E.coli BL21(DE3)::2SD::kan/pKD46 E.coli BL21(DE3)::3SD::kan/pKD46、E.coli BL21(DE3)::4SD::kan/pKD46接种到无抗性LB培养基培养,取菌液在无抗性LB平板上划线,将单菌落分别对应点在卡那霉素抗性平板和氨苄青霉素抗性平板上,在卡那霉素抗性平板上生长而氨苄青霉素抗性平板上不生长的菌株即为E.coli BL21(DE3)::1SD::kan、E.coli BL21(DE3)::2SD::kan、E.coliBL21(DE3)::3SD::kan E.coli、BL21(DE3)::4SD::kan。制备E.coli BL21(DE3)::1SD::kan、E.coli BL21(DE3)::2SD::kan、E.coli BL21(DE3)::3SD::kan、E.coli BL21(DE3)::4SD::kan电转感受态细胞,转化pCP20质粒,涂布于氨苄青霉素抗性、氯霉素抗性双抗性LB培养基平板上,获得含pCP20质粒的阳性转化子,将其接种到无抗性LB培养基中培养,消除pCP20质粒,获得BL21(+1SD)和BL21(+2SD)、BL21(+3SD)、BL21(+4SD)重组大肠杆菌,其中,各重组大肠杆菌中,距离DacA的初始密码子ATG最近的SD序列与DacA的初始密码子ATG的距离均为0bp。其中,抗性基因消除后BL21::1SD、BL21::2SD、BL21::3SD、BL21::4SD菌株中的同源片段理论上分别长517、523、529、535bp,菌落PCR扩增验证结果与理论值一致。BL21::1SD、BL21::2SD菌株在Kan、氨苄青霉素(Amp)平板上均不生长,在无抗性LB平板上正常生长。
实施例2基因整合SD序列至羧肽酶DacA的初始密码子ATG不同距离对E.coli胞外生产重组蛋白的影响
本实施例中测定分析了基因整合1SD序列至羧肽酶DacA的初始密码子ATG不同距离对E.coli胞外生产重组蛋白的影响。本实施例中,以GFP为模式蛋白进行验证。分别构建距离DacA的初始密码子ATG分别为0、2、4、6、8、10个碱基的重组菌株,获得重组菌株BL21(+1SD)、BL21(+1SD)-2、BL21(+1SD)-4、BL21(+1SD)-6、BL21(+1SD)-8、BL21(+1SD)-10,并分别转化重组质粒pET28a-gfp。如图2所示,距离0个碱基时BL21(+1SD)-gfp重组GFP的胞外分泌水平最高,其胞外GFP荧光值可到2.6*105A.U.·L·g-1,所以优选距离为0个碱基。
实施例3重组大肠杆菌胞外生产重组绿色荧光蛋白
本实施例中测定分析了基因整合对E.coli胞外生产GFP的影响。
将重组质粒pET28a-gfp分别转化至出发菌株BL21、实施例1构建的BL21(+1SD)和BL21(+2SD)菌株中,构建重组突变株BL21-gfp,BL21(+1SD)-gfp和BL21(+2SD)-gfp。将重组突变株BL21-gfp,BL21(+1SD)-gfp和BL21(+2SD)-gfp在LB培养基中37℃培养8h,然后按1%(v/v)的接种量接种到TB培养基中,37℃培养至OD600=0.8,加入终浓度为1mmol·L-1IPTG,25℃诱导表达。
测定绿色荧光蛋白荧光的含量。如图3所示,BL21(+1SD)-gfp和BL21(+2SD)-gfp突变菌株的胞外GFP荧光值分别为2.8×105和2.6×105A.U.·L·g-1,而对照菌株BL21-gfp仅为2.1×105A.U.·L·g-1。与对照菌株相比,整合1SD序列后重组突变株的胞外GFP产量提高至1.3倍,整合2SD序列后重组突变株的胞外GFP产量提高至1.2倍,整合1SD序列和2SD序列后,有利于GFP的胞外生产。
同时,还通过SDS-PAGE分析了突变菌株BL21(+1SD)-gfp、BL21(+2SD)-gfp和对照菌株BL21-gfp的重组GFP的胞外浓度,结果表明,BL21(+1SD)-gfp的胞外重组GFP的浓度高于BL21(+2SD)-gfp,BL21(+2SD)-gfp的胞外重组GFP的浓度高于BL21-gfp,再次验证了引入不同数目SD序列后重组GFP的胞外分泌水平提高。
实施例4重组大肠杆菌胞外生产重组淀粉酶
本实施例中将重组质粒pET28a-amyk分别转化到出发菌株BL21、实施例1构建的BL21(+1SD)和BL21(+2SD)突变菌株中,获得重组突变菌株BL21-amyk、BL21(+1SD)-amyk和BL21(+2SD)-amyk。将BL21-amyk、BL21(+1SD)-amyk和BL21(+2SD)-amyk分别在LB培养基中37℃培养8h,然后按1%(v/v)的接种量接种到TB培养基中,37℃培养至OD600=0.8,加入终浓度为1mmol·L-1IPTG,25℃诱导表达。发酵结束后,离心、收集发酵上清液,检测发酵上清液中的淀粉酶活力,分析在E.coli中引入不同数目SD序列对细胞外重组淀粉酶产生的影响。
如图4(a)所示,诱导20h时,突变株BL21(+1SD)-amyk和BL21(+2SD)-amyk的胞外重组淀粉酶酶活力分别达到1765.0U·g-1和1386.6U·g-1,而对照菌株(BL21-amyk)仅为879.4U·g-1,可见,整合1SD、2SD序列后可以显著提高重组淀粉酶胞外生产水平。通过SDS-PAGE分析1SD、2SD序列整合对细胞外重组淀粉酶表达的影响(图4(b)),在突变菌株中发现了对应于AmyK分子量(62.8kDa)的明显蛋白质条带(图4(b)中箭头所指处),再次表明整合1SD、2SD序列可以促进重组淀粉酶分泌到胞外。
实施例5引入不同数目SD序列对E.coli细胞膜渗透性的影响
疏水性荧光探针N-苯基-1-萘胺(NPN)可用作外膜完整性的指示剂。NPN在水溶液中具有低荧光量子产率,但在生物膜的疏水环境中强烈发出荧光。通常,NPN通过外膜的脂多糖层从E.coli中排出,可以在膜完整性受损的点进入E.coli。因此,可以用荧光强度表征细胞膜外膜渗透性。将NPN加入到对照菌株BL21、实施例1构建的重组菌株BL21(+1SD)、BL21(+2SD)的细胞悬浮液时反应剧烈,测得荧光值如图5所示。相对于对照菌株BL21,突变株BL21(+1SD)、BL21(+2SD)的荧光强度由1.5×104A.U.分别提高至1.75×104和1.7×104A.U.。这说明引入1SD序列后提高了E.coli外膜的渗透性,对细胞膜外膜完整性有一定影响。
实施例6引入不同数目SD序列对E.coli的α-半乳糖苷酶酶的影响
根据绘制的对硝基酚浓度标准曲线,从而进行α-半乳糖苷酶酶活测定。实施例1中构建的启动子改造菌株BL21(+1SD)、BL21(+2SD)的酶活力分别为107.8、82.2U·g-1(图6所示),而对照菌株D,D-羧肽酶酶活力仅为50.9U·g-1。
实施例7引入不同数目SD序列对羧肽酶表达量的影响
羧肽酶属于膜上蛋白,通过测定膜上羧肽酶酶活,进而表征启动子改造对羧肽酶表达量的影响。启动子改造后重组菌株细胞膜上的D,D-羧肽酶酶活力显著提高,实施例1中构建的启动子改造菌株BL21(+1SD)、BL21(+2SD)的酶活力分别为18.4、16.7U·g-1,而对照菌株D,D-羧肽酶酶活力仅为9.4U·g-1。在E.coli中启动子改造提高了D,D-羧肽酶DacA的表达,这为胞内可溶性肽聚糖增加及重组蛋白胞外分泌水平提高的主要原因。
对比例:
设计引物并PCR扩增含有Kan抗性基因的用于替换目的基因dacD的同源片段,采用实施例1中记载的敲除dacA或dacB的方法,敲除大肠杆菌中的基因dacA,获得突变株BL21-ΔdacD。将重组质粒pETDuet-gfp转化至突变株BL21-ΔdacD后,突变菌株的胞外GFP荧光值为88.1A.U.·L·g-1,而对照菌株BL21-pETDuet-gfp为87.5A.U.·L·g-1。可以看出,D,D-羧肽酶dacD基因敲除对大肠杆菌胞外蛋白生产几乎没有效果。
以上实施例以采用绿色荧光蛋白(GFP)和淀粉酶为模式重组蛋白,证明了本发明方法可显著提高胞外蛋白的含量。整合1SD序列和2SD序列后,GFP胞外荧光值分别提高1.2和1.3倍;整合1SD序列和2SD序列后,淀粉酶胞外酶活力分别提高了2.0和1.6倍。
以上仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
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<120> 提高大肠杆菌重组蛋白胞外分泌水平的方法
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Claims (10)
1.一种提高大肠杆菌重组蛋白胞外分泌水平的方法,其特征在于,通过同源重组的方法将包含1-4个SD序列的基因片段整合至编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子与DacA编码基因之间。
2.根据权利要求1所述的方法,其特征在于:编码所述SD序列的核苷酸序列如SEQ IDNo.1所示。
3.根据权利要求1所述的方法,其特征在于:距离DacA初始密码子ATG最近的SD序列与初始密码子ATG序列的距离为0-20bp。
4.根据权利要求1所述的方法,其特征在于:所述大肠杆菌是E.coli BL21、E.coliJM109、E.coli DH5α、E.coli Rosetta、E.coli TOP10、E.coliM110或E.coliS110。
5.根据权利要求1-4中任一项所述的方法,其特征在于,包括以下步骤:
(1)设计并合成含有抗性基因及待整合基因上下游同源臂的整合框;所述待整合基因包括编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子,所述上下游同源臂中包括1-4个SD序列;
(2)将所述整合框电转化至含有质粒载体的大肠杆菌电转化感受态细胞,同源重组后筛选出基因整合的大肠杆菌,并消除抗性基因。
6.一种重组大肠杆菌,其特征在于,所述重组大肠杆菌中,编码D-丙氨酰-D-丙氨酸羧肽酶DacA的启动子和DacA编码基因之间整合有1-4个SD序列。
7.根据权利要求6所述的重组大肠杆菌,其特征在于,编码所述SD序列的核苷酸序列如SEQ ID No.1所示。
8.根据权利要求6所述的重组大肠杆菌,其特征在于,距离DacA初始密码子ATG最近的SD序列与初始密码子ATG的距离为0-20bp。
9.权利要求6所述的重组大肠杆菌在重组蛋白胞外生产中的应用。
10.根据权利要求9所述的应用,其特征在于:所述重组蛋白包括淀粉酶、绿色荧光蛋、胶原蛋白、果糖基转移酶、甲基对硫磷水解酶、过氧化氢酶和葡萄糖氧化酶中的一种或几种。
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