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The swine leukocyte antigen (SLA) genes in pigs were the important immune gene group in antigen presentation, and studing on SLA could provide the references for the prevention of some infectious diseases. Earlier studies found that SLA-1-632-TPK gene in ToPigs pig had a deleted base in its coding sequence (a single base "C" was lost in 632 bp from the 5' end of the SLA-1-632-TPK gene), which lead to frameshift mutation. In order to correct the SLA-1-632-TPK gene, two pairs of gene-correction's primers were designed to correct the gene by the splicing overlap extension PCR (SOE-PCR) in template of recombinant plasmid of SLA-1-632-TPK/pMD18-T. Firstly,the 5'and 3'ends of SLA-1-632-TPK gene were amplified, respectively, then both of them were spliced and amplified to form an intact SLA-1-632-TPK gene. After detected by agarose electrophoresis, the interest of the product was further cloned into pMD19-T Simple vector. The positive clones were screened by colony PCR and then sequenced. The result showed that the 5'and 3' ends of the SLA-1-632-TPK gene were all amplified successfully by SOE-PCR, with the products of about 650 and 590 bp, which were consistent with the theoretical value of 648 and 585 bp, respectively. After spliced, the intact sequence of SLA-1-632-TPK gene was obtained with the product of about 1 200 bp, which was close to the theoretical value of 1 223 bp. The colony PCR result showed that the corrected gene was successfully inserted into pMD19-T Simple vector . After the sequence was analyzed by GENETYX version 9.0, it was shown that the nucleotide "C" in 632 bp numbered from the 5'end of the gene was added and the SLA-1-632-TPK gene was coded correctly. In this study, the SLA-1-632-TPK was corrected successfully, and the recombinant plasmid SLA-1-TPK/pMD19-T was constructed, which would lay a foundation to further study the protein expression and associated function of SLA-1-TPK. 相似文献
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猪白细胞抗原（swine leukocyte antigen,SLA）在猪免疫系统中起递呈抗原作用,对其展开研究可为猪相关传染病的预防提供依据。研究发现,托佩克猪SLA-1-632-TPK基因编码序列发生碱基缺失（距离SLA-1-632-TPK基因5'端632 bp处丢失1个碱基"C"）,导致移码突变。为矫正SLA-1-632-TPK基因,设计2对矫正引物,以原重组质粒SLA-1-632-TPK/pMD18-T为模板,利用剪切重叠延伸PCR（splicing overlap extention PCR,SOE-PCR）技术分别扩增SLA-1-632-TPK 5'和3'端,之后进行拼接,最后扩增全序列从而矫正目的基因,并进一步连接pMD19-T Simple载体,通过单菌落PCR筛选阳性克隆并测序,并通过GENETYX version 9.0软件对所测序列进行分析。结果显示,SOE-PCR成功扩增得到5'和3'端片段,大小约为650和590 bp,与理论设计值大小（648和585 bp）接近,经过拼接以后,得到全长约1 200 bp,与理论设计值1 223 bp接近。菌落PCR结果显示,矫正基因成功克隆入pMD19-T Simple载体。序列分析结果显示,托佩克猪SLA-1-632-TPK基因距离5'端632 bp处丢失的碱基"C"得到矫正并正确编码。本研究成功矫正了SLA-1-632-TPK基因,并构建其重组质粒SLA-1-TPK/pMD19-T,为下一步研究SLA-1-TPK蛋白表达和相关功能奠定基础。 相似文献
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本研究利用改进SOE-PCR技术构建肝靶向穿膜肽（HTPP）与家蝇天蚕素（MDC）融合基因并对其分子特征进行了预测和分析.结果表明：成功融合了HTPP与MDC,并构建了HTPP-MDC融合基因的克隆重组质粒HTPP-MDC/pMD20-T.PCR和KpnⅠ/Hind Ⅲ双酶切结果显示获得与预期大小一致的基因片段,测序结果显示获得的基因序列没有发生突变,与预期完全一致.分子特征分析表明,该融合基因编码60个氨基酸,分子量为6 516.2 Da,理论等电点为 9.31,二级结构主要由α-螺旋、无规则卷曲、延伸链和β-转角组成.研究结果为HTPP-MDC后续的功能研究奠定了基础,同时也为应用SOE-PCR技术构建融合基因提供了有益借鉴. 相似文献
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为构建长链CpG ODN DNA,将合成的含有CpG的GTCGTT核心序列以TT间隔开后重复3次,并在3′端添加polyG结构,而后在其两翼人为添加Xba Ⅰ的黏性末端(简称为A链)后合成与其互补的互补链(简称为B链),通过退火形成CpG ODN DNA单体,以DNA连接酶将CpG单体连接为多聚体后再以A链和B链作为引物,利用SOE-PCR技术对poly CpG ODN DNA进行长链扩增,分别将600～700 bp片段克隆到pMD-18T载体中,并获得相应的重组子pUC44、pUC26及pUC30.序列分析结果表明:3个重组子与理论上产生的含有10,11,13个CTAGATCGT(CG)TTTTGT(CG)TTTTGT(CG)TTGAGGGGGGAT拷贝长链CpG ODN DNA标准序列的同源性分别为97.4%、97.9%和97.1%. 相似文献
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抗非洲猪瘟病毒单链抗体的构建、表达及活性鉴定 总被引：1，自引：0，他引：1
制备抗非洲猪瘟病毒（ASFV）猪源单链抗体（ScFv）,并对其生物学活性进行鉴定,筛选出具有ASFV反应活性的猪源单链抗体（ScFv）,为ASFV的诊断提供新的材料。采集ASFV感染康复猪的外周血,分离淋巴细胞,提取淋巴细胞总RNA,以此为模板通过PCR扩增得到猪源IgG重链可变区与轻链可变区基因,利用SOE-PCR技术扩增拼接得到猪源ScFv基因;构建pET-30a-ScFv表达载体,进行蛋白的表达与纯化,用ELISA和IFA鉴定ScFv抗体的反应活性。结果显示,成功扩增出猪源ScFv（VH-VLκ、VH-VLλ）基因,鉴定到1株与ASFV存在反应活性的ScFv（VH-VLλ11）抗体。结果表明,成功筛选到1株与ASFV存在反应活性的ScFv（VH-VLλ11）抗体,为ASFV诊断和防控提供了新型原材料。 相似文献
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为原核表达猪嗜血支原体(M.haemosuis) ORF9蛋白,本研究根据GenBank登录的AJ504999的ORF9基因序列,采用重叠延伸PCR技术(SOE-PCR),选择大肠杆菌偏爱的密码子,设计并合成4对寡核苷酸片段,人工合成ORF9基因.将其克隆于表达载体pET-32a中构建重组表达质粒pET-ORF9,转化大肠杆菌BL21(DE3)中,IPTG诱导表达.经SDS-PAGE和western blot鉴定表明,重组蛋白的分子量约为34 ku. 相似文献
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为探究绵羊肺炎支原体（Mycoplasma ovipneumoniae,Mo）pcDNA3.1-TBP30-Hsp70融合表达质粒对小鼠细胞免疫应答影响,本试验构建了绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒。用已构建的pMD19T-P30和pMD19-Hsp70质粒为模板,采用基因定点突变（SDM）原理设计引物,应用SOE-PCR扩增目的基因片段,并将其定向克隆至表达载体pcDNA3.1（+）,构建重组质粒pcDNA 3.1（+）-TBP30和融合重组质pcDNA3.1（+）-TBP30-Hsp70。使用pcDNA3.1-TBP30、pcDNA3.1-TBP30-Hsp70、pcDNA3.1（+）和Elution Buffer对小鼠进行免疫,应用ELISA试剂盒检测小鼠血清中细胞因子白细胞介素-2（IL-2）、IL-4、干扰素-γ（INF-γ）分泌水平。结果显示,pcDNA3.1-TBP30-Hsp70酶切后可见大小分别约为1 413 bp的目的基因片段和5 400 bp的载体条带。与空白对照组和pcDNA3.1（+）组相比,免疫重组质粒组均可引起小鼠血清中细胞因子INF-γ、IL-2和IL-4分泌水平的增强,与空白对照组和pcDNA3.1（+）组相比差异显著或极显著（P<0.05;P<0.01）;而空白对照组和空质粒组之间差异不显著（P>0.05）;免疫pcDNA3.1-TBP30和pcDNA3.1-TBP30-Hsp70组小鼠血清IL-2、INF-γ和IL-4终分泌量增加,表明重组质粒组可刺激小鼠血清中IL-2、INF-γ和IL-4的变化,并且在时间上都呈现出先增多后减少的规律。本试验结果表明,重组质粒pcDNA3.1（+）-TBP30-Hsp70免疫小鼠后,IL-2和INF-γ分泌水平的升高,增强了机体的细胞免疫功能,进而调节机体细胞免疫影响T细胞和巨噬细胞的分泌,从而提高机体细胞免疫能力;IL-4分泌水平升高,促进机体Th2向Th1分化,维持Th1的优势状态,增强了机体的细胞免疫功能。本试验结果为绵羊肺炎支原体基因工程疫苗的研制提供了参考依据。 相似文献
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[目的]构建联合表达气肿疽梭菌的鞭毛蛋白与分泌蛋白神经氨酸酶的方法,用以制备气肿疽生物工程亚单位疫苗.[方法]根据已公布的气肿疽梭菌FliA（C）和NanA序列设计引物,联合使用SOE-PCR与TD-PCR技术,建立融合基因FliA（C）-NanA的扩增方法.[结果]试验成功扩增出了融合基因FliA（C）-NanA.[结论]SOE-PCR方法无需在引物设计中加入酶切位点进行连接,降低了试验成本,且2个基因间未加入其他核酸序列,避免了表达出影响免疫效果的蛋白;TD-PCR的应用节省了试验时间,避免了筛选退火温度的繁琐步骤,解决了基因丢失的问题. 相似文献