动物乳腺特异表达载体的构建及其表达特性研究

为了研制动物乳腺生物反应器,用中国奶山羊β-乳球蛋白基因的5′、3′-侧翼区和β-酪蛋白基因的第1内含子构建动物乳腺特异表达载体p205C3.细胞表达试验结果显示,p205C3载体能够指导lacZ基因在SV40 T基因转化的山羊乳腺上皮细胞中表达,不能指导该基因在COS-1细胞和山羊成纤维细胞中表达.将人激肽释放酶(hKLK1) cDNA克隆入p205C3载体,用获得的重组载体p205C3KLK1注射哺乳期小鼠,然后取其心、肝、脾、肺、肾、脑、肌肉、胰腺和乳汁,酶活性测定结果显示,乳汁中的酶活性高达255.65 U·mL-1,其他组织中的酶活性与正常小鼠无差异.结果表明p205C3载体不仅能有效地指导外源基因在山羊乳腺细胞和小鼠乳腺中表达,而且表达具有较好的组织特异性,可以用于动物乳腺生物反应器的研制.     

甜味蛋白Brazzein基因酵母表达系统的建立

根据甜味蛋白Brazzein基因成熟区的氨基酸序列,结合毕赤酵母的密码子偏爱性以及Brazzein蛋白质结构的相关研究,对甜味蛋白Brazzein基因进行改造,使表达的目的蛋白中包含3个突变氨基酸(29Asp→Lys,31His→Ala,41Glu→Lys)。采用重叠PCR(SOE-PCR)合成目的基因并克隆到pMD18-T载体,经测序鉴定,序列正确的质粒用EcoRⅠ和XbaⅠ双酶切。将回收得到的目的基因连接到经同样双酶切处理的pGAPZαA载体上,经PCR、酶切和测序鉴定,证明已成功构建pGAPZαA-Bra真核表达载体。将构建好的重组载体pGAPZαA-Bra用AvrⅡ线性化,电转进入巴斯德毕赤酵母中。筛选ZeocinTM阳性克隆菌,进行蛋白表达,SDS-PAGE结果表明蛋白表达成功。     

Brazzein基因的合成及pGAPZαA-Bra表达载体的构建

[目的]为Brazzein基因在酵母SMD1168中转化和表达奠定基础。[方法]利用重叠延伸PCR(SOE-PCR)合成Brazzein基因,将其连接到pMD18-T载体上,构建克隆载体pMD18-T-Bra。分别用XhoI和XbaI限制内切酶对克隆质粒pMD18-T-Bra和酵母表达载体pGAPZαA进行酶切,并在T4DNA连接酶作用下,将回收的目的基因连接到pGAPZαA载体上,构建重组质粒pGAPZαA-Bra。[结果]通过PCR扩增获得了约188 bp的Brazzein类似物的编码序列,将其克隆到pMD18-T质粒,用XhoI和XbaI双酶切后,连接到pGAPZαA载体,成功构建了重组表达载体pGAPZαA-Bra。Brazzein基因其中各碱基未发生突变,整个表达载体阅读框正确无误。[结论]利用基因工程的方法生产Brazzein是可行的。     

转人乳铁蛋白基因山羊乳腺上皮细胞的体外诱导表达

[目的]为了探索山羊乳腺上皮细胞体外诱导表达的技术体系,评价乳腺特异性载体效率及外源蛋白在细胞水平的表达情况。[方法]用含5mg/L胰岛素、5mg/L催乳素、1mg/L氢化可的松的DMEM/F12培养液对转染人乳铁蛋白基因的山羊乳腺上皮细胞进行体外诱导培养,每6h收集1次上清液。上清液浓缩后分别用SDS-PAGE和Westernblot检测外源蛋白的表达情况。[结果]诱导培养液中有目的蛋白表达,分子量约为42kD。[结论]该研究所用的山羊乳腺细胞诱导表达方法能够诱导山羊乳腺上皮细胞表达外源基因,这为人乳铁蛋白基因的异源表达与乳腺生物反应器的研究奠定了基础。     

乳腺特异表达载体缺失片段的鉴定与修复

通过对pBC1中的乳腺特异表达启动子成分进行分析,鉴定了位于β酪蛋白启动子上游两个EcoRⅠ位点(-89~-94和-120~-125)之间的缺失片段,31bp的缺失使得该载体中的一个乳控盒元件(-112~-141)和一个乳腺因子识别序列(-92~-102)遭到破坏,并使得位于TATA框上游的所有启动子成分发生了位置改变。通过与山羊β酪蛋白启动子序列进行比较,设计并合成了缺失片段,经一系列的酶切和连接处理,使得该缺失得到了修复,修复后的载体具有所有乳蛋白表达需要的启动子调控原件。本试验结果为利用修复后的乳腺特异高效表达载体进行乳腺生物反应器的研究奠定了分子生物学基础。     

山羊乳腺特异性表达慢病毒载体的构建与验证

[目的]构建山羊乳腺特异性表达尿激酶原突变体的重组慢病毒载体,证明其表达的有效性。[方法]将劳氏肉瘤病毒增强子／启动子、复制缺陷型艾滋病病毒（HIV-1）的5’端长重复序列（LTR）、HIV-1ψ卫包装信号、HIVRev反应元件、山羊B-casein调控序列、pro—UKM13、△U3／3’LTR依次连接,构建乳腺特异性表达的慢病毒载体;通过体外转染人乳腺癌细胞系（MCF-7）、仓鼠卵巢细胞（CHO）和泌乳山羊乳腺注射证明其表达有效性。[结果]构建的山羊乳腺特异性表达载体酶切鉴定是正确的;利用该载体转染细胞,采用溶圈法和Westernblot检测证实了其表达的有效性;慢病毒载体注射到泌乳山羊的乳腺,在乳汁中也检测到了尿激酶原的表达。[结论]为在转基因动物的乳腺中表达尿激酶原突变体奠定了基础。     

甜味蛋白Brazzein基因的合成及在毕赤酵母中的表达

用连接PCR合成出甜味蛋白Brazzein的基因,克隆到PUC57质粒中测序后,用EcoR I和Not I双酶切,连接到pPIC9k,电转化毕赤酵母GS 115,经MD和MM平板筛选,获得His^ Mut^ 重组子。0．5％甲醇诱导表达96h后,对发酵液和细胞进行SDS—PAGE分析,在发酵液中没有蛋白带出现,在细胞裂解液中出现相对分子质量约6．0kd的特异性甜味蛋白条带,与预期的相对分子质量大小相符。用BCA蛋白定量试剂盒测定,Brazzein在毕赤酵母中的表达量可达329mg／L。     

山羊α-乳清蛋白乳腺特异性表达载体的构建 及核供体细胞的转染

为了构建α-乳清蛋白乳腺特异性表达载体及提供山羊乳腺生物反应器α-乳清蛋白核移植供体细胞,提取乳腺组织中的总RNA,采用RT-PCR技术获得α-乳清蛋白基因cDNA,测序酶切后连接乳腺特异性表达载体pBCⅠ-Neo,并对重组载体进行酶切测序鉴定;取重组正确的载体酶切线性化后利用脂质体介导转染至山羊胎儿成纤维细胞,通过G418筛选培养转染了α-乳清蛋白乳腺特异性表达载体的山羊胎儿成纤维细胞。结果显示:经过RT-PCR特异性扩增克隆出α-乳清蛋白基因;克隆的基因碱基组成序列经测序与预期完全一致,α-乳清蛋白基因片段正向插入乳腺特异性表达载体;重组载体在山羊胎儿成纤维细胞中稳定转染,转入α-乳清蛋白乳腺特异性表达载体的山羊胎儿成纤维细胞生长迅速,仍保持原有的细胞生长形态。成功构建了α-乳清蛋白乳腺特异性表达载体,获得稳定转染的山羊胎儿成纤维细胞可用于核移植。     

GPAM基因过表达载体的构建及其在奶牛乳腺上皮细胞中的表达

线粒体3-磷酸甘油酰基转移酶(GPAM,glycerol-3-phosphate acyltransferase)是一种具有编码蛋白质功能的基因,位于牛的26号染色体上。GPAM酶参与催化三酰基甘油和磷脂生物合成反应的第一步,继而对甘油三酯的量进行调控,是生物体内一个必不可少的酶。试验以中国荷斯坦牛RNA反转录成cDNA,继而PCR扩增出GPAM片段,并连入pBI-CMV3载体,构建了pBI-CMV3-GPAM真核表达载体,并验证了其在奶牛乳腺上皮细胞中的表达情况。结果表明:成功构建了牛GPAM真核表达载体,并在体外奶牛乳腺上皮细胞中高效表达。     

利用SRY基因鉴定山羊胎儿成纤维细胞的性别

根据GenBank中已发表的山羊SRY基因和β-乳球蛋白基因序列设计2对PCR引物,对来自3个1月龄山羊胎儿成纤维细胞的基因组DNA进行PCR扩增,分别以公羊、母羊的基因组DNA为阳性和阴性对照,同时对PCR产物进行序列测定和同源性分析。结果表明,阳性对照和GFF1细胞系有2条扩增带,阴性对照、GFF2和GFF3细胞系只有1条扩增带;序列测定结果表明,PCR扩增产物为SRY基因,利用该方法准确鉴定出1个雄性细胞系和2个雌性细胞系。     

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.     

萨能奶山羊乳腺组织5-HTR2基因的克隆与序列分析

以容易获取并具代表性的乳腺组织——奶山羊乳腺为载体,进行哺乳动物乳腺组织中5-HTR2基因的克隆,为下一步研究奶牛乳腺组织中5-HTR基因的分布及生物学特性提供理论基础.根据GenBank其他物种5- HT受体- 2(5 - HTR2)基因序列的同源序列,对奶山羊5-HTR2基因设计1对克隆引物,从奶山羊乳腺组织提取总...     

西农萨能羊乳腺脂肪酸合酶基因exon 9-15的克隆与序列分析

【目的】山羊FAS基因exon 9-15编码的乙酰/丙二酸单酰基转移酶（acetyl-CoA and malonyl-CoA transacylases,AT/MT）区域对山羊乳短、中链脂肪酸的合成起重要调控作用。本研究针对西农萨能羊乳腺FAS基因exon 9-15进行克隆和序列分析。【方法】以处于泌乳期28 d的西农萨能羊乳腺组织mRNA反转录的cDNA为模板,通过RT-PCR方法首次扩增出西农萨能羊乳腺FAS基因exon 9-15的cDNA序列全长及exon 8 的3′端和exon 16 的5′端部分cDNA序列（GenBank收录号为DQ 915966）,并对其进行了同源性分析和功能预测。【结果】克隆片段全长1 449 bp,其中包括exon 9-15共1 388 bp、exon 8的3′端9 bp和exon 16 的5′端52 bp,编码483个氨基酸,包含编码的AT/MT区域951 bp（454～1404 nt）;西农萨能羊乳腺FAS基因exon 9-15与牛（NM_001012669）,人（NM_004104）,大鼠（NM_017332）和鸡（NM_205155）核苷酸序列相似度分别为95%、85.7%、82.7%、73.2%,氨基酸相似度分别为92.9%、77.7%、82.3%、64.7%;exon 10较其它物种缺失1个氨基酸。【结论】克隆获得西农萨能羊FAS基因片段全长1 449 bp,外显子9-15大小分别为463、185、190、95、135、204和116 bp;核苷酸及氨基酸序列与牛相应序列的同源性均高于其它物种;AT/MT区域的活性位点在各物种间均为高度保守的丝氨酸,但西农萨能羊exon 10较其它物种缺失1个氨基酸,这可能对AT/MT区域的空间构象及其生理功能产生重要影响。本研究为西农萨能羊FAS基因cDNA全长克隆以及基因功能研究奠定了重要基础。     

西农萨能羊FAS基因shRNA序列筛选及其腺病毒载体的构建

山羊脂肪酸合酶(Fatty acid synthase,FAS)是脂肪酸合成的关键酶,对乳腺短、中链脂肪酸合成起重要调控作用。设计了shRNA-5544、shRNA-5936s、hRNA-6132 3条针对FAS基因不同区域的小发夹RNA(Short hairpin RNA,shRNA)及一条阴性对照序列shRNA-NC,并构建表达这4条shRNA序列的入门载体及其靶基因与红色荧光蛋白基因的融合表达载体,二者共转染HEK 293细胞进行有效序列筛选,结果显示shRNA-5544和shRNA-5936序列具有明显的干扰效果。在LR Clonase-Ⅱ重组酶作用下,分别将pENTR/CMV-GFP/U6-shRNA-5544、pENTR/CMV-GFP/U6-shRNA-5936及pENTR/CMV-GFP/U6-shRNA-NC入门载体与腺病毒骨架载体pAd/PL-DEST进行LR重组,经氨苄青霉素及氯霉素抗性筛选后成功获得3个重组腺病毒载体,ScaⅠ酶切鉴定及测序分析证实所构建的重组腺病毒载体中插入序列与设计序列一致。重组腺病毒载体经PacⅠ酶切线性化后,利用Lipofectamine 2000转染HEK 293细胞,10~12 d后收集病毒,在HEK 293细胞中反复扩增3次后,获得高滴度的重组腺病毒,利用TCID50法测定重组腺病毒滴度分别为6×108PFU/mL(表达shRNA-5544序列)、5×108PFU/mL(表达shRNA-5936序列)及6×108PFU/mL(表达shRNA-NC序列),为进一步在原代培养的山羊乳腺上皮细胞中进行FAS基因的RNA干扰研究奠定基础。     

体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究

[目的]探讨供体细胞类型、移植胚胎发育阶段、数量及部位对山羊转基因克隆效率的影响。[方法]利用体细胞核移植技术将转染人乳铁蛋白基因hLF的山羊胎儿成纤维细胞(GFF)和乳腺上皮细胞(GMGE)移植到MII期去核卵母细胞内,经电融合、激活、体外培养后,2~8细胞期克隆胚被移植到同期发情山羊的输卵管内,囊胚被移植到子宫角内。[结果]GFF与GMGE的妊娠率相近(输卵管移植妊娠率分别为26.47%及20.00%);在GFF,输卵管移植的妊娠率与子宫角内移植妊娠率接近(分别为26.47%及25.00%),输卵管移植胚胎平均数为21.2组的妊娠率显著高于5.93组和9.64组(40.00%及26.67%,21.43%)。[结论]供体细胞类型、移植胚胎的发育阶段及移植部位对山羊转基因克隆效率的影响不大,但对于输卵管移植,受体羊移植胚胎数量对妊娠率有明显的影响。此外,该研究还提示了利用成年羊乳腺上皮细胞制作转基因动物的可行性。     

体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究(英文)

[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn’t be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.     

体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究(英文)

[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.     

Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer

[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.