CN113881618B - 一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用 - Google Patents
一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用,其中所述重组枯草芽孢杆菌以枯草芽孢杆菌为出发菌株,敲除了内切‑β‑1,3‑1,4‑葡萄糖苷酶基因的编码区,并整合表达α‑S1‑酪蛋白基因、β‑酪蛋白基因和氯霉素抗性基因,其中所述α‑S1‑酪蛋白基因的核苷酸序列如SEQ ID NO.4所示、所述β‑酪蛋白基因的核苷酸序列如SEQ ID NO.5所示。本发明以枯草芽孢杆菌的bglS基因为基础,利用同源重组的方法,将α‑S1‑酪蛋白基因和β‑酪蛋白基因以及氯霉素抗性筛选标记基因串联在一起,替换bglS基因编码区,利用麦芽汁诱导即可获得了高效分泌酪蛋白的重组枯草芽孢杆菌基因工程菌,可作为饲料添加剂,在断奶仔畜、畜禽和水产动物生产中具有极高的应用前景。
Description
技术领域
本发明属于遗传工程技术领域,具体涉及一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用。
背景技术
乳蛋白含有几乎全部必需氨基酸,也被称为“全蛋白”,是新生幼仔最具营养价值的蛋白质。常乳中,酪蛋白在牛乳和猪乳中分别占总蛋白的80%和60%。乳中酪蛋白主要分为3种类型:α-S-酪蛋白、β-酪蛋白和κ-酪蛋白,其中α-S-酪蛋白是牛乳中主要的酪蛋白(α-S1-酪蛋白约占总酪蛋白的38%、α-S2-酪蛋白约占总酪蛋白的10%),β-酪蛋白在牛乳中的含量仅次于α-S1-酪蛋白,约占总酪蛋白的35%。α-S1-酪蛋白和β-酪蛋白不仅含有全部必需氨基酸,在新生幼仔胃肠道蛋白酶消化降解后,蛋白裂解还可以产生多种生物活性肽。这些活性肽包括:阿片肽、免疫调节肽、抗菌肽、DNA合成促进肽、降压肽、酪蛋白磷酸肽等,对幼仔的消化、营养和免疫等发挥调节作用。
枯草芽孢杆菌(Bacillus subtilis)是一种重要的益生菌种,不含内毒素和无致病性,被美国FDA认定为“Generally Regarded as Safe”(GRAS)级别益生菌种,在中国也已作为益生菌被批准为饲料添加剂。枯草芽孢杆菌在普通碳源培养基中生长良好,在工业生产条件下保持较好的活力,无显著的密码子偏爱性,适于作为“细菌工厂”生产外源重组蛋白。
内切-β-1,3-1,4-葡萄糖苷酶(EC3.2.1.73)是一种β-葡聚糖水解酶,产该酶的细菌主要是芽胞杆菌。该酶能够特异性水解β-1,3-1,4-葡聚糖中与β-1,3糖苷键相连的β-1,4糖苷键,能够促进谷物中β-1,3-1,4-葡聚糖的水解。在利用LB液体培养基培养枯草芽孢杆菌时,该酶表达量很低。β-葡聚糖可以作为诱导物诱导内切-β-1,3-1,4-葡萄糖苷酶(bglS)基因的大量表达,在枯草芽孢杆菌人工培养过程中,bglS基因可以被培养基的谷物成分如麦芽汁(含有大量的β-葡聚糖)诱导表达。
发明内容
本发明的目的在于提供了一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用,本发明通过研究发现内切-β-1,3-1,4-葡萄糖苷酶(bglS)基因对于人工培养的枯草芽孢杆菌属于非必需基因,并且可被β-葡聚糖或培养基的谷物成分如麦芽汁诱导表达,因此以枯草芽孢杆菌的bglS基因为基础,利用同源重组的方法,将α-S1-酪蛋白(α-S1-CN)基因和β-酪蛋白(β-CN)基因以及氯霉素抗性(CmR)筛选标记基因串联在一起,替换bglS基因编码区,利用麦芽汁诱导即可获得了高效分泌酪蛋白的重组枯草芽孢杆菌基因工程菌。
本发明的目的之一在于提供了一种重组枯草芽孢杆菌,所述重组枯草芽孢杆菌以枯草芽孢杆菌为出发菌株,敲除了其中的内切-β-1,3-1,4-葡萄糖苷酶基因的编码区,并整合表达α-S1-酪蛋白基因、β-酪蛋白基因和氯霉素抗性基因,其中所述α-S1-酪蛋白基因的核苷酸序列如SEQ ID NO.4所示、所述β-酪蛋白基因的核苷酸序列如SEQ ID NO.5所示。
进一步地,所述整合表达为将所述α-S1-酪蛋白基因、β-酪蛋白基因和氯霉素抗性基因依次串联在一起,通过同源重组替换枯草芽孢杆菌的内切-β-1,3-1,4-葡萄糖苷酶基因编码区。
进一步地,所述α-S1-酪蛋白基因前端连接有如序列表SEQ ID NO.3所示的信号肽序列,所述β-酪蛋白基因和氯霉素抗性基因之间连接有如SEQ ID NO.6所示的转录终止子序列。
进一步地,所述氯霉素抗性基因包括:依次连接的如SEQ ID NO.7所示的氯霉素抗性基因启动子序列以及如SEQ ID NO.8所示的氯霉素抗性基因编码区序列。
进一步地,所述重组枯草芽孢杆菌以枯草芽孢杆菌1A1菌株为出发菌株。
本发明的目的之二在于提供了上述重组枯草芽孢杆菌的构建方法,包括:
步骤1、将如SEQ ID NO.1所示的内切-β-1,3-1,4-葡萄糖苷酶基因的上游同源臂、SEQ ID NO.3所示的信号肽序列、SEQ ID NO.4所示的α-S1-酪蛋白基因、SEQ ID NO.5所示的β-酪蛋白基因、SEQ ID NO.6所示的转录终止子序列、SEQ ID NO.7所示的氯霉素抗性基因启动子序列、SEQ ID NO.8所示的氯霉素抗性基因编码区序列以及SEQ ID NO.2所示的内切-β-1,3-1,4-葡萄糖苷酶基因的下游同源臂顺序连接,形成同源重组表达盒;
步骤2、将所述同源重组表达盒连入表达载体中,形成整合表达载体;
步骤3、将所述整合表达载体转化至出发菌株枯草芽孢杆菌中,筛选阳性克隆并进行检测;
步骤4、对阳性克隆进行培养,诱导阳性克隆表达α-S1-酪蛋白和β-酪蛋白串联蛋白。
进一步地,步骤1的同源重组表达盒构建过程中,先依次合成内切-β-1,3-1,4-葡萄糖苷酶基因的上游同源臂片段、信号肽序列-α-S1-酪蛋白-β-酪蛋白-转录终止子串联基因片段、氯霉素抗性基因片段和内切-β-1,3-1,4-葡萄糖苷酶基因的下游同源臂片段,然后利用重叠延伸PCR的方法将四个片段连接,得到同源重组表达盒。
进一步地,步骤2中将同源重组表达盒通过无缝拼接连入E.coli-Bacillussubtilis穿梭表达载体pHY300PLK的XbaⅠ位点,形成整合表达载体。
进一步地,步骤3中将整合表达载体采用电转化法转化至出发菌株枯草芽孢杆菌1A1菌株中,采用抗生素/无抗生素培养基交替筛选法筛选阳性克隆。
进一步地,所述抗生素/无抗生素培养基交替筛选法具体为:取待筛选的枯草芽孢杆菌,采用含氨苄青霉素的LB琼脂培养基培养枯草芽孢杆菌1代,再用无抗生素的LB琼脂培养基培养10代,再用含氯霉素的LB琼脂培养基培养8代,再用无抗生素的LB琼脂培养基培养10代,得到阳性克隆。
进一步地,步骤3中筛选得到阳性克隆后,通过PCR扩增检测同源重组表达盒整合效果,通过PCR扩增和序列测定确定重组序列的准确性。
进一步地,步骤4中采用含1%麦芽糖的无抗生素LB培养基对阳性克隆进行培养,并诱导阳性克隆表达α-S1-酪蛋白和β-酪蛋白串联蛋白。
本发明的目的之三在于提供了上述重组枯草芽孢杆菌在发酵生产乳酪蛋白和/或制备饲料添加剂中的应用。
与现有技术相比,本发明的有益效果是:
(1)本发明通过研究发现bglS基因对于人工培养的枯草芽孢杆菌属于非必需基因,并且可被β-葡聚糖或培养基的谷物成分如麦芽汁诱导表达,因此以枯草芽孢杆菌的bglS基因为基础,利用同源重组的方法,将α-S1-CN基因和β-CN基因以及氯霉素抗性(CmR)筛选标记基因串联在一起以替换bglS基因编码区,即可获得了高效分泌酪蛋白的重组枯草芽孢杆菌基因工程菌,仅利用麦芽汁进行体外诱导培养即可大量表达α-S1-酪蛋白和β-酪蛋白串联蛋白,产量最高可达693.5mg/L。
(2)本发明将所述重组枯草芽孢杆菌的发酵代谢产物作为添加剂(按1.5%w/w比例)饲喂断奶子鼠可显著增重,增重率相对出发菌株提高7.38%,因此本发明制备得到的重组枯草芽孢杆菌作为饲料添加剂,在断奶仔畜、畜禽和水产动物生产中具有极高的应用前景。
(3)本发明建立了一套基于大肠杆菌-枯草芽孢杆菌穿梭载体电转化和同源重组的方法,对后续利用诱导型枯草芽孢杆菌非必需基因表达外源重组蛋白具有重要理论和实际意义。
附图说明
图1为本发明实施例1中同源重组双交换技术原理示意图;
图2为本发明实施例2中整合表达载体转化和阳性克隆筛选示意图;
图3为本发明实施例2中同源重组表达盒整合效果的PCR扩增和琼脂糖凝胶电泳检测结果;
图4为本发明实施例3中重组枯草芽孢杆菌发酵液中α-S1-酪蛋白和β-酪蛋白串联蛋白含量;
图5为本发明实施例4中重组枯草芽孢杆菌的发酵代谢产物作为饲料添加剂的增重效果。
具体实施方式
下面将结合本发明中的实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1用于同源重组双交换的整合表达载体的构建
本发明采用同源重组双交换的方法将α-S1-CN基因和β-CN基因以及CmR筛选标记基因串联在一起以替换bglS基因编码区,技术原理示意图如图1所示,其中整合表达载体构建方法如下:
(1)设计和人工合成用于替换枯草芽孢杆菌(Bacillus subtilis)bglS基因(genebank ID:937470)编码区的上游同源臂序列bglSup 400bp,如序列表SEQ ID NO.1所示。
(2)设计和人工合成用于替换枯草芽孢杆菌bglS基因(gene bank ID:937470)编码区的下游同源臂序列bglSdown 400bp,如SEQ ID NO.2所示。
(3)设计和人工合成连有信号肽序列(GenBank ID:K01985.1)的α-S1-CN(genebank ID:M38641.1)和β-CN(gene bank ID:M55158.1)串联基因,其中信号肽序列如SEQ IDNO.3所示,α-S1-CN基因的序列如SEQ ID NO.4所示,β-CN基因的序列如SEQ ID NO.5所示。
(4)设计和人工合成α-S1-CN和β-CN串联基因的转录终止子序列(克隆自pHY300PLK的AmpR抗性基因转录终止子),如SEQ ID NO.6所示。
(5)利用本领域常规的重叠延伸PCR方法或人工合成的方法获得信号肽-α-S1-CN-β-CN-转录终止子串联序列。
(6)设计和人工合成氯霉素抗性CmR基因序列,其中包括如SEQ ID NO.7所示的CmR基因启动子序列以及如SEQ ID NO.8所示的CmR基因编码区序列。
将以上(1)、(2)、(5)和(6)步骤中获得的4个片段bglSup、信号肽-α-S1-CN-β-CN-转录终止子串联序列、CmR和bglSdown利用重叠延伸PCR进行片段连接。获得的融合基因片段bglSup-α-S1-CN-β-CN-CmR-bglSdown为替换bglS基因编码区的同源重组表达盒(含有上下游同源臂,之间串联α-S1-CN基因、β-CN基因以及CmR基因序列。)
将同源臂序列通过本领域常规的无缝拼接方法,连入E.coli-Bacillus subtilis穿梭表达载体pHY300PLK的XbaⅠ位点,形成整合表达载体,将其命名为pHY300PLK-ABC。
实施例2整合表达载体转化和阳性克隆筛选
1、制备枯草芽孢杆菌1A1感受态细胞:利用LB液体培养基于37℃培养枯草芽孢杆菌1A1菌株(Bacillus subtilis 1A1,购于美国俄亥俄州立大学杆菌保藏中心BGSC)至OD600达到0.8,然后将菌体用与LB液体培养基等体积的预冷HSMG缓冲液(含1mM HEPES,1mMMgCl2,0.5M山梨醇,10%甘油)洗涤4~5次,获得感受态细胞。
2、整合表达载体pHY300PLK-ABC经电转化方法转入感受态枯草芽孢杆菌,其中电转化条件为:将以上菌体用HSMG缓冲液高密度悬浮(约浓缩50倍),再加入十分之一体积的pHY300PLK质粒,冰浴10min。混合物加入预冷的电转杯中,电击条件:2.5kV,400Ω,25μF。电击后的菌体迅速冰浴制冷,转至SOC复苏培养基中于37℃培养3~4h,之后再转至含氨苄青霉素LB琼脂培养基上培养。
3、应用含Cm抗生素/无抗生素培养基交替筛选法筛选转化子阳性克隆,具体步骤如下:
pHY300PLK载体表达含有氨苄青霉素抗性基因(AmpR),应用含氨苄青霉素LB琼脂培养基培养枯草芽孢杆菌1代,用于筛选含有pHY300PLK载体转化子的阳性克隆。该阳性克隆中存在既含有pHY300PLK载体又发生同源重组的菌株,以备下一步筛选。
再采用无抗生素的LB液体培养基培养枯草芽孢杆菌10代,用于筛选pHY300PLK-ABC载体消除的同源重组枯草芽孢杆菌阳性克隆。
接着再采用含氯霉素(Cm)培养基培养8代,用于进一步筛选消除pHY300PLK载体并发生同源重组具有CmR的重组枯草芽孢杆菌阳性克隆。
最后再用无抗生素的LB固体培养基培养枯草芽孢杆菌10代,以筛选稳定同源重组的克隆。挑选平板中长出的单菌落,采用本领域常规的PCR扩增方法检测同源重组表达盒整合效果。其中整合表达载体转化和阳性克隆筛选示意图如图2所示。
PCR扩增引物序列如下:
F,5'CGTATGAATGCACGAAGA3';(如SEQ ID NO.9所示)
R,5'CACCTGCCGTTGATCACT3'(如SEQ ID NO.10所示)。
扩增片段大小3282bp,检测结果如图3所示。通过PCR扩增和序列测定确定重组序列的准确性。获得的菌株即为稳定发生同源重组的目的菌株。
实施例3发酵生产乳酪蛋白
将实施例2筛选得到的重组枯草芽孢杆菌阳性克隆进行发酵培养并诱导表达,其中发酵培养基为:含1%麦芽汁的无抗生素LB液体培养基。
接种和培养条件:将重组枯草芽孢杆菌阳性克隆按5%接种量转入发酵培养基,于37℃培养28~32h。利用麦芽汁诱导重组枯草芽孢杆菌表达α-S1-酪蛋白和β-酪蛋白串联蛋白。
应用本领域常规的ELISA检测试剂盒检测发酵液中酪蛋白含量,并以未采用麦芽汁诱导作为对照。发酵液中α-S1-酪蛋白和β-酪蛋白串联蛋白的含量随时间变化如图4所示,结果显示,本发明制备得到重组枯草芽孢杆菌发酵液中酪蛋白含量最高可达693.5mg/L。
实施例4重组枯草芽孢杆菌的应用以及断奶子鼠增重实验
本实施例提供了所述重组枯草芽孢杆菌在作为饲料添加剂,以增加饲喂动物体重的应用,具体如下:
将重组枯草芽孢杆菌用于饲料发酵,其中培养基组成:按80%玉米粉+15%豆粕+5%麦麸比例混合配制干物质含量达到15%的液体培养基,再添加1%麦芽汁,发酵36h,获得枯草芽孢杆菌发酵代谢产物。并以不含整合表达载体pHY300PLK-ABC的出发菌株(枯草芽孢杆菌1A1菌株)作为对照。
将上述重组枯草芽孢杆菌发酵代谢产物作为添加剂(按1.5%w/w比例)饲喂断奶子鼠(雌鼠),从第三周龄起(第22日龄,断奶第一天)饲喂至第8周龄末(第56日龄),并统计子鼠增重率,结果如图5所示。结果显示,,采用本发明所述的重组枯草芽孢杆菌的发酵代谢产物作为添加剂可显著增加饲喂动物的体重,相较于出发菌株(WT),其体重增长率提高了7.38%。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,都应涵盖在本发明的保护范围之内。
序列表
<110> 长江大学
<120> 一种分泌乳酪蛋白的重组枯草芽孢杆菌及其构建方法和应用
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<400> 2
aagaaaatcc aaacctacat tgagcgggag tatgagcaca agctcacaag tgacgagctg 60
ctgtatttaa ccattcacat agaaagggta gttaaacaag cataatgaga gcgctgacat 120
ttgtgtttcc ttgtgttcac tttttcttac attcacatat gaaaatggta ggattgttac 180
tgataaagca ggcaaaacct aaattgcaat gagtgcggat catctctgtc tgtgctgatg 240
gtaatttagg tttttatttt tttcagaggg aagatgatga tagttacagg attcaagtta 300
gtaagattcg atattatcat tattttgacc gatgttccct tttaaaagaa tcatgtaaga 360
tcaacataga aaacgctttc aatgaaaggg gaatgccaat 400
<210> 3
<211> 90
<212> DNA
<213> 枯草芽孢杆菌(Bacillus subtilis)
<400> 3
gtgggtttag gtaagaaatt gtctgttcgt gtcgctgctt cgtttatgag tttatcaatc 60
agcctgccag gtgttcaggc tgctgaaggt 90
<210> 4
<211> 642
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaaacttc tcatccttac ctgtcttgtg gctgttgctc ttgccaggcc taaacatcct 60
atcaagcacc aaggactccc tcaagaagtc ctcaatgaaa atttactcag gttttttgtg 120
gcactttttc cagaagtgtt tggaaaggag aaggtcaatg aactgagcaa ggatattggg 180
agtgaatcaa ctgaggatca agccatggaa gatattaagc aaatggaagc tgaaagcatt 240
tcgtcaagtg aggaaattgt tcccaatagt gttgagcaga agcacattca aaaggaagat 300
gtgccctctg agcgttacct gggttatctg gaacagcttc tcagactgaa aaaatacaaa 360
gtaccccagc tggaaattgt tcccaatagt gctgaggaac gacttcacag tatgaaagag 420
ggaatcgatg cccaacagaa agaacctatg ataggagtga atcaggaact ggcctacttc 480
taccctgagc ttttcagaca attctaccag ctggatgcct atccatctgg tgcctggtat 540
tacgttccac taggcacaca atacactgat gccccatcat tctctgacat ccctaatccc 600
attggctctg agaacagtga aaagactact atctcactgt gg 642
<210> 5
<211> 675
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaaggtcc tcatccttgc ctgcctggtg gctctggccc ttgcaagaga gctggaagaa 60
ctcaatgtac ctggtgagat tgtggaaagc ctttcaagca gtgaggaatc tattacacgc 120
atcaataaga aaattgagaa gtttcagagt gaggaacagc agcaaacaga ggatgaactc 180
caggataaaa tccacccctt tgcccagaca cagtctctag tctatccctt ccctggaccc 240
atccataaca gcctcccaca aaacatccct cctcttactc aaacccctgt ggtggtgccg 300
cctttccttc agcctgaagt aatgggagtc tccaaagtga aggaggctat ggctcctaag 360
cacaaagaaa tgcccttccc taaatatcca gttgagccct ttactgaaag ccagagcctg 420
actctcactg atgttgaaaa tctgcacctt cctctgcctc tgctccagtc ttggatgcac 480
cagcctcacc agcctcttcc tccaactgtc atgtttcctc ctcagtccgt gctgtccctt 540
tctcagtcca aagtcctgcc tgttccccag aaagcagtgc cctatcccca gagagatatg 600
cccattcagg cctttctgct gtaccaggag cctgtactcg gtcctgtccg gggacccttc 660
cctattattg tctaa 675
<210> 6
<211> 235
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 60
aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 120
ttttcgttcc actgagcgtc agacccctta ataagatgat cttcttgaga tcgttttggt 180
ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggagg ttttt 235
<210> 7
<211> 98
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgaagcaata atggaggaat ggttgacttc aaaacaaata aattatataa tgacctttgt 60
gtgaaatatt gcagaagctt acataaggag gaactact 98
<210> 8
<211> 651
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgaacttta ataaaattga tttagacaat tggaagagaa aagagatatt taatcattat 60
ttgaaccaac aaacgacttt tagtataacc acagaaattg atattagtgt tttataccga 120
aacataaaac aagaaggata taaattttac cctgcattta ttttcttagt gacaagggtg 180
ataaactcaa atacagcttt tagaactggt tacaatagcg acggagagtt aggttattgg 240
gataagttag agccacttta tacaattttt gatggtgtat ctaaaacatt ctctggtatt 300
tggactcctg taaagaatga cttcaaagag ttttatgatt tatacctttc tgatgtagag 360
aaatataatg gttcggggaa attgtttccc aaaacaccta tacctgaaaa tgctttttct 420
ctttctatta ttccatggac ttcatttact gggtttaact taaatatcaa taataatagt 480
aattaccttc tacccattat tacagcagga aaattcatta ataaaggtaa ttcaatatat 540
ttaccgctat ctttacaggt acatcattct gtttgtgatg gttatcatgc aggattgttt 600
atgaactcta ttcaggaatt gtcagatagg cctaatgact ggcttttata a 651
<210> 9
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgtatgaatg cacgaaga 18
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cacctgccgt tgatcact 18
Claims (6)
1.一种重组枯草芽孢杆菌,其特征在于,所述重组枯草芽孢杆菌以枯草芽孢杆菌1A1为出发菌株,敲除了其中的内切-β-1,3-1,4-葡萄糖苷酶基因的编码区,并整合表达α-S1-酪蛋白基因、β-酪蛋白基因和氯霉素抗性基因,其中所述α-S1-酪蛋白基因的核苷酸序列如SEQ ID NO. 4所示、所述β-酪蛋白基因的核苷酸序列如SEQ ID NO. 5所示;
所述整合表达为将所述α-S1-酪蛋白基因、β-酪蛋白基因和氯霉素抗性基因依次串联在一起,通过同源重组替换枯草芽孢杆菌的内切-β-1,3-1,4-葡萄糖苷酶基因编码区;
所述α-S1-酪蛋白基因前端连接有如序列表SEQ ID NO. 3所示的信号肽序列,所述β-酪蛋白基因和氯霉素抗性基因之间连接有如SEQ ID NO. 6所示的转录终止子序列;
所述氯霉素抗性基因包括:依次连接的如SEQ ID NO. 7所示的氯霉素抗性基因启动子序列以及如SEQ ID NO. 8所示的氯霉素抗性基因编码区序列。
2.如权利要求1所述的一种重组枯草芽孢杆菌的构建方法,其特征在于,所述方法包括:
步骤1、将如SEQ ID NO. 1所示的内切-β-1,3-1,4-葡萄糖苷酶基因的上游同源臂、SEQID NO. 3所示的信号肽序列、SEQ ID NO. 4所示的α-S1-酪蛋白基因、SEQ ID NO. 5所示的β-酪蛋白基因、SEQ ID NO. 6所示的转录终止子序列、SEQ ID NO. 7所示的氯霉素抗性基因启动子序列、SEQ ID NO. 8所示的氯霉素抗性基因编码区序列以及SEQ ID NO. 2所示的内切-β-1,3-1,4-葡萄糖苷酶基因的下游同源臂顺序连接,形成同源重组表达盒;
步骤2、将所述同源重组表达盒连入表达载体中,形成整合表达载体;
步骤3、将所述整合表达载体转化至出发菌株枯草芽孢杆菌中,筛选阳性克隆并进行检测;
步骤4、对阳性克隆进行培养,诱导阳性克隆表达α-S1-酪蛋白和β-酪蛋白串联蛋白。
3. 根据权利要求2所述的构建方法,其特征在于,步骤2中将同源重组表达盒通过无缝拼接连入E.coli-Bacillus subtilis穿梭表达载体pHY300PLK的Xba Ⅰ位点,形成整合表达载体。
4.根据权利要求2所述的构建方法,其特征在于,步骤3中将整合表达载体采用电转化法转化至出发菌株枯草芽孢杆菌1A1菌株中,采用抗生素/无抗生素培养基交替筛选法筛选阳性克隆。
5.根据权利要求2所述的构建方法,其特征在于,步骤4中采用含1%麦芽糖的无抗生素LB培养基对阳性克隆进行培养,并诱导阳性克隆表达α-S1-酪蛋白和β-酪蛋白串联蛋白。
6.如权利要求1所述的重组枯草芽孢杆菌在发酵生产乳酪蛋白和/或制备饲料添加剂中的应用。
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