CN106893742A - 一种动物全身性表达重组人溶菌酶的方法 - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
一种动物全身性表达重组人溶菌酶的方法,其特征在于第一步:人溶菌酶BAC克隆选择Genome Systems公司的GenBank号为RP11‑72P21作为目标hLZ BAC克隆;第二步:冗余基因CPSF6的去除:为避免冗余基因CPSF6对hLZ基因的干扰,通过重组方法去除冗余基因CPSF6,获得去除冗余基因的人溶菌酶pBAC‑hLZ‑Neo。本发明与已有技术相比,具有能在身体的多个部位都有表达的优点。各组织器官中表达的重组人溶菌酶均有生物活性;肠道中表达的重组人溶菌酶能极显著增加双歧杆菌的数量(P<0.001),同时极显著降低沙门氏菌的数量(P<0.001)。
Description
技术领域
本发明涉及一种通过重组而获得新的基因的技术。
背景技术
溶菌酶,又称胞壁质酶或N-乙酰胞壁质聚糖水解酶,是一种能水解致病菌中黏多糖的碱性酶。主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡糖之间的β-1,4糖苷键,使细胞壁不溶性黏多糖分解成可溶性糖肽,导致细胞壁破裂内容物逸出而使细菌溶解。溶菌酶还可与带负电荷的病毒蛋白直接结合,与DNA、RNA、脱辅基蛋白形成复盐,使病毒失活。因此,该酶具有抗菌、消炎、抗病毒等作用。
目前,与人类生活密切相关的溶菌酶主要鸡蛋清溶菌酶,广泛来源于禽类的蛋清,其来源广泛,纯化工艺简单,是认识最为清楚,也是唯一得到工业化应用的溶菌酶,但存在过敏性和活性低等缺点。
人溶菌酶(hLZ)是溶菌酶家族的成员之一,作为人体的非特性免疫物质之一,人溶菌酶在全身粘膜组织中广泛表达,包括泪液、唾液、鼻粘液、尿液、消化液、汗水及人的乳汁,发挥着抗菌消炎的重要功能。同时作为一种免疫因子,人溶菌酶尤其在中性粒细胞和巨噬细胞的分泌物中发挥作用。
相比蛋清溶菌酶,人溶菌活性最高,为鸡蛋清溶菌酶的3倍,有更高的热稳定性,无过敏性,临床应用时无刺激性及副作用等突出优点,开发潜力巨大。但是,目前人溶菌酶来源渠道狭小,主要来源于母乳和血液的提纯,限制了其产业化应用。
通过基因工程技术,重组人溶菌酶已在酵母、植物和转基因动物乳腺中等系统中获得表达,其中以转基因动物乳腺表达最为突出。1994年Mega等首先进行了转基因小鼠乳腺表达人溶菌酶的研究,他们利用alpha s1 酪蛋白启动子来诱导溶菌酶的表达,表达量为0.2到0.7克/升;1998年,Mega等的研究结果进一步表明转基因小鼠表达人溶菌酶的奶样具有同标准溶菌酶相同的活性。这些结果暗示着溶菌酶的转基因研究在乳品加工业有着巨大的应用前景。2006年,国内首次利用体细胞克隆的方法获得了乳腺特异性表达重组人溶菌酶的转基因牛。虽然如此,已有技术所获得的人溶菌酶主要是乳腺表达,而不能实现身体多个部位的表达。
发明内容
本发明的发明目的在于提供一种能在动物体多个组织中全身性表达重组人溶菌酶的方法。
本发明是这样实现的,
hLZ BAC克隆,GenBank号为RP11-72P21,含有人溶菌酶基因组序列和完整的调控序列(来源于Genome Systems公司);
SW102菌株、PL452质粒(来源于美国国立卫生研究院生物资源分部);
Taq DNA聚合酶购于大连TaKaRa公司,引物合成及序列测定由上海生工完成;兔抗人溶菌酶多克隆抗体购自US Biological公司,人溶菌酶ELISA检测试剂盒购于BiomedicalTechnologies公司;酶切、连接、回收、转化、PCR扩增等常规实验操作步骤采用《分子克隆(第三版)》的技术,
第一步:人溶菌酶BAC克隆选择Genome Systems公司的GenBank号为RP11-72P21作为目标hLZ BAC克隆,该hLZ BAC克隆含有155kb 5’调控区—CPSF6冗余基因,4.8kb hLZ基因组序列和6kb 3’调控区;
第二步:冗余基因CPSF6的去除:为避免冗余基因CPSF6对hLZ基因的干扰,通过重组方法去除冗余基因CPSF6,具体步骤如下:
(1)hLZ BAC克隆通过电转染方法转入SW102菌株(该菌株已转入了Red重组系统所需要的exo蛋白、bet蛋白、gam蛋白);SW102菌株是美国国立卫生研究院生物资源分部提供,
(2)选择CPSF6冗余基因外显子1为重组置换区域,设计同源臂,以pBR322-loxP-Neo质粒设计引物扩增loxP-Neo基因片段,同时将同源臂和引物连接,获得包含如下重组引物的PCR产物,F-Neo:5’-TCT CTG AGG ATG CAG AAA CAA GAC ATT GTT TTT GGT CAT ACG CCG TTC ACA GAA TGC CCG CCG CCT CCA TCC AGT CTA TT -3’;R-Neo:5’-TTA TCT GTG GCT CCA AAA GTC AAA GGA CTT GTT GCA AAT GAG CCC GCA CCT CAG TCG CCTGTC GCC GCATAC ACT ATT CT-3’,其中,CG CCT CCA TCC AGT CTA TT和GTC GCC GCA TAC ACT ATT CT为引物,其他为同源臂,
(3)重组引物所得到的PCR产物,切胶回收后,电击转入含有含hLZ BAC的SW102菌株中,发生重组,获得去除冗余基因的人溶菌酶pBAC-hLZ-Neo。
本发明与已有技术相比,所获得的该人溶菌酶pBAC-hLZ-Neo经过试验验证,具有能在身体的多个部位都有表达的优点。
附图说明
图1表达载体pBAC-hLZ-Neo构建和鉴定;
(A) 利用重组方法,通过Neo抗性的置换去除冗余基因CPSF6;
(B) PCR鉴定阳性克隆,发生重组的菌株(编号1-10)均能扩增出2.5kb条带,NC为阴性对照;
(C) 测序法进一步确认重组的发生,箭头所指为重组发生的起始位置;
图2 转基因小鼠的分子生物学检测;
(A) PCR检测,阳性小鼠14、18、22、30可扩增637bp的条带,阴性对照NC无条带;
(B) Southern blot检测. 阳性质粒P和阳性小鼠14、18、22、30可杂出3.3kb条带,阴性对照NC无条带;
(C) RT-PCR检测转基因小鼠重组人溶菌酶的转录,重组人溶菌酶在心H、肝Li、脾Sp、肺Lu、肾K、胃St、肠I、肌肉Mu等组织中均有RNA水平的转录;
图3 重组人溶菌酶表达检测与活性分析;
(A) Western blot检测重组人溶菌酶在转基因小鼠各个组织器官中的表达,重组人溶菌酶在脾Sp、肺Lu、肾K、胃St、肠I、肝Li等组织中均有蛋白水平的表达。
(B)Western blot检测重组人溶菌酶在转基因小鼠血清中的表达. 阳性对照PC上扬两为1 μg,阴性对照NC为普通小鼠血清,1-7为转基因小鼠的血清(上样量为10 μL)。
(C) 琼脂平板法测定血清中表达的重组人溶菌酶对溶壁微球菌的抑菌活性,阳性对照PC上样量为1 μg,阴性对照NC为普通小鼠血清,1-7为转基因小鼠的血清(上样量为10μL)。
具体实施方式
现结合实施例对本发明做进一步详细描述:
第一步:人溶菌酶BAC克隆选择Genome Systems公司的GenBank号为RP11-72P21作为目标hLZ BAC克隆,该hLZ BAC克隆含有155kb 5’调控区—CPSF6冗余基因,4.8kb hLZ基因组序列和6kb 3’调控区,
hLZ BAC克隆的提取采用试剂盒(NucleoBond BAC 100 Kit)提供的方法和步骤进行,粗提hLZ BAC克隆则是在加完P1、P2和P3等溶液后,直接用0.7倍体积的异丙醇沉淀,粗提的hLZ BAC克隆用于SW102菌株的电击转化:
第二步:冗余基因CPSF6的去除:为避免冗余基因CPSF6对hLZ基因的干扰,通过重组方法去除冗余基因CPSF6,步骤如下:
(1)hLZ BAC克隆通过电转染方法转入SW102菌株(该菌株已转入了Red重组系统所需要的exo蛋白、bet蛋白、gam蛋白);SW102菌株是美国国立卫生研究院生物资源分部提供,
(2)选择CPSF6冗余基因外显子1为重组置换区域,设计同源臂,以pBR322-loxP-Neo质粒设计引物扩增loxP-Neo基因片段,同时将同源臂和引物连接,获得包含如下重组引物的PCR产物,F-Neo:5’-TCT CTG AGG ATG CAG AAA CAA GAC ATT GTT TTT GGT CAT ACG CCG TTC ACA GAA TGC CCG CCG CCT CCA TCC AGT CTA TT -3’;R-Neo:5’-TTA TCT GTG GCT CCA AAA GTC AAA GGA CTT GTT GCA AAT GAG CCC GCA CCT CAG TCG CCTGTC GCC GCATAC ACT ATT CT-3’,其中,CG CCT CCA TCC AGT CTA TT和GTC GCC GCA TAC ACT ATT CT为引物,其他为同源臂,
pBR322-loxP-Neo质粒是由pBR322质粒(pBR322为常规质粒,实验室保留)和PL452质粒(由国外同行赠与,具体网址为http://recombineering.ncifcrf. gov/Plasmid.asp#PL452)杂合而成,扩增PL452质粒的loxP-Neo片段,通过PstI酶切位点,插入到pBR322质粒中的抗性AMP基因内(破坏了AMP基因),从而构建该质粒,
(3)重组引物所得到的PCR产物(该PCR产物含两端各带60 bp的同源臂(或称重复序列,即与BAC的特定序列相重复,相当于可以铆钉在BAC的具体位置上),以及LoxP-Neo基因片段),切胶回收后,电击转入含有hLZ BAC的SW102菌株中,发生重组,获得去除冗余基因的人溶菌酶pBAC-hLZ-Neo:
具体步骤如下:
A、 SW102菌株的电转化感受态制备
a.初始培养,用枪头挑取SW102菌株单克隆,加入盛有5ml LB培养基的灭菌管中,32℃,220rpm,过夜培养;
b.扩大培养,将过夜培养液按大约1:70的比例转移到盛有50ml LB培养基的灭菌三角瓶中,32℃,220rpm,待OD600达到0.4时,停止培养;
c.将菌液平均分成2份,第一份于42℃(严格)水浴,220rpm,振荡孵育15min;第二份为对照,32℃水浴,220rpm,振荡孵育15min;
d.将2份菌液在冰水浴中快速冷却10min以上,之后于4℃,5000rpm离心10min;
e.弃上清,重新将管置于冰水浴中,加入1ml冰上预冷的ddH2O,轻轻转动重悬,再加入10ml预冷的ddH2O,快速倒转几次,4℃,5000rpm离心5min;
f.弃上清,重新将管置于冰水浴中,加入1ml冰上预冷的10%甘油+90% ddH2O,轻轻转动重悬,再加入10ml预冷的10%甘油+90% ddH2O,快速倒转几次,4℃,5000rpm离心5min;
g.弃上清,加入100-200 μL预冷的10%甘油+90% ddH2O,重悬沉淀;
h.分装,每0.5ml的小管中加入40μl,液氮速冻后-80℃保存,备用,该保存、备用物即为感受态细胞;
B、感受态细胞与PCR产物的电转化
a.取40 μl含有hLZ BAC克隆的感受态细胞与5μl 的PCR产物混合,然后将混合物转移至冰浴充分的0.2 cm电击杯中,注意不能形成气泡;
b.将电击杯周围的水擦干,置于电击槽中,启动细菌的电击程序;
c.听见“噗”的一声后,电击转化结束,向电击杯中加入0.8ml SOC培养基,用枪吸出,置于1.5ml 离心管,32℃水浴振荡恢复培养2小时;
d.涂布于含有相应的抗生素的LB板上,32℃培养箱中培养过夜;
e.PCR和Cracking法鉴定阳性克隆;
C、带有同源臂的PCR长片段引物设计
带有同源臂的长片段引物一般结构为:在普通PCR引物的5’端各带上大约50-60bp的同源序列,并可在同源臂和普通引物之间引入酶切位点等序列,修饰hLZ BAC克隆同源臂的设计如下:选取目标序列的5’端紧邻约50-60bp序列作为5’同源臂,加到一条普通引物的5’端;选取目标序列的3’端紧邻约50-60bp序列,取其反向互补序列加到另一条普通引物的5’端,作为3’同源臂;
D、阳性克隆的PCR和测序法鉴定
使用P-Neo杂合引物(F:5’-CGC CAG AGA CAA CCT ACT GT-3’和R:5’- GTC GCC GCATAC ACT ATT CT -3’)鉴定重组后的阳性克隆菌株,如果发生了重组则可扩增出2.5 kb的条带,未发生重组的菌株不能扩增出条带(图1.B);
PCR产物进一步测序,和原序列比对后发现,重组后产生的DNA置换位点和目标设计完全一致(图1.C);人溶菌酶全身性表达载体pBAC-hLZ-Neo构建完成。
检测例1:转基因小鼠模型的建立及检测
A、显微注射制备转基因小鼠
转基因小鼠制备过程参见《小鼠胚胎操作实验指南》(纳吉等著,2004,科学出版社),制备流程如下:
B、转基因小鼠的PCR检测
取2-3周龄转基因小鼠的尾巴组织样,提取基因组(常规动物DNA提取方法)。以提取的基因组为模板,用P-hLZ-637引物(5’-TTA TAC ACA CGG CTT TAC-3’和5’-CAG CAT CAGCGA TGT TAT CT-3’)在阳性小鼠中可以扩增出637bp的片段,以普通小鼠(非转基因小鼠)基因组为模板的PCR反应为阴性对照,以载体pBAC- hLZ-Neo为模板的反应为阳性对照。扩增条件为94℃,30sec;60℃,30sec;72℃,60sec;35个循环,1.0%琼脂糖电泳观察结果。
PCR结果显示,共获得4只F0代转基因小鼠,编号为:14,18,22,30(图2.A,)。
C、转基因小鼠的Southern blotting 检测
将PCR检测为阳性的F0代5只转基因小鼠,绞尾部组织并提取基因组,取大约10μg用EcoRI内切酶消化,以EcoRI酶切的野生型小鼠基因组为阴性对照,EcoRI酶切的pBAC- hLZ-Neo注射载体片段为阳性对照,杂交探针是引物P-hLZ-637的PCR产物,长度为637bp,由地高辛(DIG)标记。将各种样品低压慢速电泳,采用碱转移方法将DNA从琼脂糖转移至尼龙膜,将尼龙膜有DNA的一面朝内,卷成筒状放入杂交管中,加入预杂交液,于65℃预杂交不短于1小时,加入经过加热变性的DIG标记的探针,65℃杂交12-16小时,洗膜后用保鲜膜包好,放进暗盒中,暗室中压上X光片,观察结果。
Southern blotting结果显示,阳性信号呈现3.3kb的杂交带(图2.B)。Southernblotting 检测结果与PCR检测结果一致,进一步验证了转基因小鼠。
检测例2:重组人溶菌酶全身性表达检测与分析
A、重组人溶菌酶全身性表达RT-PCR检测
RT-PCR是将RNA的反转录(RT)和cDNA的聚合酶链式扩增(PCR)相结合的技术。首先,通过反转录酶的作用将RNA合成 cDNA,再以cDNA为模板,扩增目的片段。
本实验应用RT-PCR检测重组人溶菌酶在小鼠各个脏器中的表达。检测组织包括:心、肝、脾、肺、肾、胃、肠、肌肉,以及泌乳时期的乳腺,使用融合引物以避免干扰,融合引物为Exon1-2-F(5’-ATC AGC CTA GCA AAC TGG AT-3’)和 Exon4-R(5’-CTC CAC AAC CTTGAA CAT AC-3’),扩增产物长度为322 bp,并以小鼠持家基因GAPDH(引物为F:5’-AGG CCGGTG CTG AGT ATG TC-3’和R:5’-TGC CTG CTT CAC CAC CTT CT-3’)为系统误差对照(530bp)。
结果表明,重组人溶菌酶在心、肝、脾、肺、肾、肠、肌肉等组织中均有RNA水平的转录(图2.C)。
、重组人溶菌酶全身性表达Western blotting 检测
采集小鼠组织和血清,其中组织包括:心、肝、脾、肺、肾、胃、肌肉、肠道等。提取小鼠组织总蛋白或直接上样小鼠血清,使用15% 的SDS-PAGE凝胶电泳,再进行Western blotting检测,设置阳性(显示14.7kDa条带)和阴性对照。
结果显示:重组人溶菌酶在心、肝、脾、肺、肾、肠、肌肉等组织,以及血清均有蛋白质水平的表达(图3.A和B),这与RT-PCR结果一致,进一步证实了重组人溶菌酶的全身性表达模式。
、血清中重组人溶菌酶表达量的ELISA检测
用ELISA法测定转基因小鼠血清中的重组人溶菌酶含量,试剂盒为美国BiomedicalTechnologies公司“Human Lysozyme ELISA Kit”。每次测量浓度每个样品做两个重复,取两个重复均值作为一次测量值,每个样品至少测浓度三次,计算浓度均值和均值标准差。
结果显示,F1代转基因小鼠18、22、30号血清中重组人溶菌酶的表达量分别为:2.09 ± 0.68、2.28 ± 0.87、2.60 ± 0.86 mg/L(表1)。
实施例3:血清中重组人溶菌酶的生物活性检测
A、琼脂平板法
分别取100uL溶壁微球菌(M. Lysodeikticus)均匀涂布于1.5%琼脂粉底肉汤培养平板上,在培养基上分别放入加有10uL血清的滤纸片,普通小鼠的血清为阴性对照(NC),阳性对照为人溶菌酶标准品,PC为1ug人溶菌酶标准品。设置4次重复,28℃培养36小时,观察抑菌圈的直径,可直观判断重组人溶菌酶的表达量及活性(图3.C)。
结果显示,F2代转基因小鼠1-7号的血清样品均能产生明显的抑菌圈,表明重组人溶菌酶具有生物活性。
、比浊度法
将溶壁微球菌在液体培养基中培养24小时,然后以5000rpm离心菌液,收集菌体。用66mM磷酸钾缓冲液冲洗菌液2次,5000rpm离心。用66mM磷酸钾缓冲液溶解菌体至OD450为0.8左右。将紫外可见光分光光度计打开预热,调制OD450。用蛋清溶菌酶制作标准曲线,各浓度为1000, 2000, 3000, 4000, 5000, 6000 and 8000 U/mL。取1uL乳汁样品,加99uL缓冲液。在光程为1cm的石英比色杯中加入2.5mL混好的菌悬液,迅速加入100uL配置好的样品。每次实验重复3次。计算抑菌活性均值和均值标准差。
结果显示,F1代转基因小鼠18、22、30号血清中重组人溶菌酶的抑菌活性分别为:685.77 ± 171.43、521.49 ± 165.57、632.87 ± 47.14 U/mL(表1)
检测例4:重组人溶菌酶在肠道中表达对菌群的影响
为检测重组人溶菌酶在肠道中表达是否会影响菌群结构,设计转基因小鼠和普通小鼠各11只,出生后于21天处死,取肠道内容物进行8种类型的菌群培养检测,8种菌群为:总厌氧菌、总好氧菌,双歧杆菌、乳酸菌等有益菌2种,梭菌、肠球菌、链球菌、大肠杆菌、沙门氏菌、葡萄球菌等有害菌6种。
结果显示,与非转基因小鼠相比,转基因小鼠肠道中的双歧杆菌数量极显著升高(P<0.001),沙门氏菌数量极显著降低(P<0.001),其它6种类型的菌群数量无显著性差异(表2)。这表明,重组人溶菌酶在肠道中表达增高了有益菌双歧杆菌的数量,同时降低了有害菌沙门氏菌的数量,这将有利于转基因小鼠的肠道健康。
表1 人溶菌酶转基因小鼠整体情况
表2 重组人溶菌酶在肠道中表达对8种菌群的影响 (单位:logCFU/g)
。
SEQUENCE LISTING
<110> 佛山科学技术学院
刘, 燊
<120> 一种动物全身性表达重组人溶菌酶的方法
<130> 2017
<160> 10
<170> PatentIn version 3.5
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<213> 人工引物
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Claims (2)
1.一种动物全身性表达重组人溶菌酶的方法,其特征在于第一步:人溶菌酶BAC克隆选择Genome Systems公司的GenBank号为RP11-72P21作为目标hLZ BAC克隆,该hLZ BAC克隆含有155kb 5’调控区—CPSF6冗余基因,4.8kb hLZ基因组序列和6kb 3’调控区;
第二步:冗余基因CPSF6的去除:为避免冗余基因CPSF6对hLZ基因的干扰,通过重组方法去除冗余基因CPSF6,具体步骤如下:
(1)hLZ BAC克隆通过电转染方法转入SW102菌株(该菌株已转入了Red重组系统所需要的exo蛋白、bet蛋白、gam蛋白);SW102菌株是美国国立卫生研究院生物资源分部提供,
(2)选择CPSF6冗余基因外显子1为重组置换区域,设计同源臂,以pBR322-loxP-Neo质粒设计引物扩增loxP-Neo基因片段,同时将同源臂和引物连接,获得包含如下重组引物的PCR产物,F-Neo:5’-TCT CTG AGG ATG CAG AAA CAA GAC ATT GTT TTT GGT CAT ACG CCGTTC ACA GAA TGC CCG CCG CCT CCA TCC AGT CTA TT -3’;R-Neo:5’-TTA TCT GTG GCTCCA AAA GTC AAA GGA CTT GTT GCA AAT GAG CCC GCA CCT CAG TCG CCT GTC GCC GCATAC ACT ATT CT-3’,其中,CG CCT CCA TCC AGT CTA TT和GTC GCC GCA TAC ACT ATT CT为引物,其他为同源臂,
(3)重组引物所得到的PCR产物,切胶回收后,电击转入含有含hLZ BAC的SW102菌株中,发生重组,获得去除冗余基因的人溶菌酶pBAC-hLZ-Neo。
2.根据权利要求1所述的动物全身性表达重组人溶菌酶的方法,其特征在于A、 SW102菌株的电转化感受态制备
a.初始培养,用枪头挑取SW102菌株单克隆,加入盛有5ml LB培养基的灭菌管中,32℃,220rpm,过夜培养;
b.扩大培养,将过夜培养液按大约1:70的比例转移到盛有50ml LB培养基的灭菌三角瓶中,32℃,220rpm,待OD600达到0.4时,停止培养;
c.将菌液平均分成2份,第一份于42℃(严格)水浴,220rpm,振荡孵育15min;第二份为对照,32℃水浴,220rpm,振荡孵育15min;
d.将2份菌液在冰水浴中快速冷却10min以上,之后于4℃,5000rpm离心10min;
e.弃上清,重新将管置于冰水浴中,加入1ml冰上预冷的ddH2O,轻轻转动重悬,再加入10ml预冷的ddH2O,快速倒转几次,4℃,5000rpm离心5min;
f.弃上清,重新将管置于冰水浴中,加入1ml冰上预冷的10%甘油+90% ddH2O,轻轻转动重悬,再加入10ml预冷的10%甘油+90% ddH2O,快速倒转几次,4℃,5000rpm离心5min;
g.弃上清,加入100-200 μL预冷的10%甘油+90% ddH2O,重悬沉淀;
h.分装,每0.5ml的小管中加入40μl,液氮速冻后-80℃保存,备用,该保存、备用物即为感受态细胞;
B、 感受态细胞与PCR产物的电转化
a.取40 μl含有hLZ BAC克隆的感受态细胞与5μl 的PCR产物混合,然后将混合物转移至冰浴充分的0.2 cm电击杯中,注意不能形成气泡;
b.将电击杯周围的水擦干,置于电击槽中,启动细菌的电击程序;
c.听见“噗”的一声后,电击转化结束,向电击杯中加入0.8ml SOC培养基,用枪吸出,置于1.5ml 离心管,32℃水浴振荡恢复培养2小时;
d.涂布于含有相应的抗生素的LB板上,32℃培养箱中培养过夜;
e.PCR和Cracking法鉴定阳性克隆;
C、带有同源臂的PCR长片段引物设计
带有同源臂的长片段引物一般结构为:在普通PCR引物的5’端各带上大约50-60bp的同源序列,并可在同源臂和普通引物之间引入酶切位点等序列,修饰hLZ BAC克隆同源臂的设计如下:选取目标序列的5’端紧邻约50-60bp序列作为5’同源臂,加到一条普通引物的5’端;选取目标序列的3’端紧邻约50-60bp序列,取其反向互补序列加到另一条普通引物的5’端,作为3’同源臂;
D、 阳性克隆的PCR和测序法鉴定
使用P-Neo杂合引物(F:5’-CGC CAG AGA CAA CCT ACT GT-3’和R:5’- GTC GCC GCATAC ACT ATT CT -3’)鉴定重组后的阳性克隆菌株,如果发生了重组则可扩增出2.5 kb的条带,未发生重组的菌株不能扩增出条带(图1.B);
PCR产物进一步测序,和原序列比对后发现,重组后产生的DNA置换位点和目标设计完全一致(图1.C);人溶菌酶全身性表达载体pBAC-hLZ-Neo构建完成。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108165567A (zh) * | 2017-12-19 | 2018-06-15 | 佛山科学技术学院 | 人溶菌酶基因的合成及其表达产物的构建方法 |
CN108251440A (zh) * | 2017-12-18 | 2018-07-06 | 宁波大学 | 缢蛏溶菌酶基因、编码蛋白及重组缢蛏溶菌酶基因工程菌的构建方法和应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007097434A (ja) * | 2005-09-30 | 2007-04-19 | Institute Of Physical & Chemical Research | 呼吸促拍症候群(rds)モデルマウス |
CN102199624A (zh) * | 2011-03-24 | 2011-09-28 | 北京济福霖生物技术有限公司 | 一种利用人工染色体在乳腺中高效生产重组蛋白的方法 |
CN104946675A (zh) * | 2015-07-02 | 2015-09-30 | 江苏敖众生物科技有限公司 | 一种鸡蛋清溶菌酶基因(rjm)的克隆及其酵母表达方法 |
-
2017
- 2017-03-17 CN CN201710162021.7A patent/CN106893742A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007097434A (ja) * | 2005-09-30 | 2007-04-19 | Institute Of Physical & Chemical Research | 呼吸促拍症候群(rds)モデルマウス |
CN102199624A (zh) * | 2011-03-24 | 2011-09-28 | 北京济福霖生物技术有限公司 | 一种利用人工染色体在乳腺中高效生产重组蛋白的方法 |
CN104946675A (zh) * | 2015-07-02 | 2015-09-30 | 江苏敖众生物科技有限公司 | 一种鸡蛋清溶菌酶基因(rjm)的克隆及其酵母表达方法 |
Non-Patent Citations (3)
Title |
---|
WATERSON RH: "Homo sapiens chromosome 12 clone RP11-72P21,WORKING DRAFT SEQUENCE,27 unordered pieces", 《GENBANK DATABASE》 * |
吴芳芳等: "转基因动物生产重组人溶菌酶的研究进展", 《中国畜牧杂志》 * |
谢泽雄等: "酵母基因组工程 ", 《中国科学:生命科学》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108251440A (zh) * | 2017-12-18 | 2018-07-06 | 宁波大学 | 缢蛏溶菌酶基因、编码蛋白及重组缢蛏溶菌酶基因工程菌的构建方法和应用 |
CN108251440B (zh) * | 2017-12-18 | 2021-04-13 | 宁波大学 | 缢蛏溶菌酶基因、编码蛋白及重组缢蛏溶菌酶基因工程菌的构建方法和应用 |
CN108165567A (zh) * | 2017-12-19 | 2018-06-15 | 佛山科学技术学院 | 人溶菌酶基因的合成及其表达产物的构建方法 |
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