【作者】 田兴华；

【导师】 吴常信； 李宁；

【作者基本信息】 中国农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 哺乳动物唾液腺是外分泌腺,也兼有部分内分泌功能。大多数分泌物可通过口腔直接到达胃部,在pH>4的情况下发挥作用;少量分泌物可直接进入血液循环发挥作用。唾液蛋白种类繁多,功能多样,往往一种蛋白有数种功能,不同蛋白有相同功能。唾液蛋白的上述特点使其具备了研制生物反应器的条件。 腮腺分泌蛋白(Parotid Secretory Protein,PSP)就是一种腮腺特异性表达的蛋白,由于它的功能与免疫相关,引起了人们的关注。为了弄清PSP的表达情况,以及它与α-AMY(α-Amylase)表达的关系,本研究根据已知的cDNA序列设计引物,利用RT-PCR、荧光定量PCR(fluorescence quantitative Polymerase Chain React,Q-PCR)对猪及小鼠的PSP、AMY在腮腺生后发育不同时期的表达情况进行了研究。结果表明,猪与小鼠的PSP在腮腺生后发育的各时期都是高效表达的,而且PSP的表达量随发育期呈动态变化;并且与AMY的变化方向一致,推测二者存在协同性表达。随后的Northern杂交和Western杂交证实了,PSP在mRNA水平和蛋白水平也是高效表达的。 对猪PSP的全长氨基酸序列进行了抗原决定簇分析,推测猪PSP有4个抗原决定簇,分别位于PSP蛋白的22-37aa,41-59aa,180-195aa以及C-端。经过比较,按其中之一合成短肽,将短肽与匙孔血蓝蛋白连接,免疫家兔,获得了抗猪PSP抗血清。从蛋白质水平对其表达情况进行了研究。在整个研究过程中,将a-唾液淀粉酶及三磷酸甘油醛脱氢酶的表达情况作为对照。研究发现PSP在猪及小鼠中都呈腮腺特异性高效表达,表达量在不同发育期呈动态变化,且始终高于GAPDH。并且在6周龄小鼠腮腺中发现了一个20KDa的蛋白,能与PSP抗体发生反应。 在第二部分中,首先利用RLM-5’RACE进行了猪PSP转录起始位点的定位,结果表明,通过RLM-5’RACE得到的序列比现有的cDNA序列的5’端长39个碱基。分析发现,现有的猪PSP基因的开放阅读框(Open Reading Fragment,ORF)有误,猪ORF的真正起点(ATG)是在现存5’端上游22个碱基处(ATGATGG)。而猪PSP基因的转录起始位点,位于该基因新的ORF起始密码子上游19个碱基处。在随后进行的PSP核心启动子预测中,分析发现猪PSP基因的启动子属于非典型启动子,不具有TATA box等经典元件;但有8个Inr在转录起始点附近,而且其中两个Inr刚好覆盖在转录起始位点上,这佐证了5’RACE结果,并且与Inr本身的特点也有很好的符合。 随后用瞬时转染法对功能性启动子进行定位。首先成功地将普通转染载体改造为启动子分析专用载体。利用一系列嵌套引物对启动子区片段进行了扩增、克隆、亚克隆,成功构建了含有不同长度5’调控区的细胞转染载体。我们利用人唾液腺癌细胞系(Acc-M)作为转染平台,我们培养了猪腮腺原代上皮细胞,将其作为Acc-M的对照和补充。我们拟用质脂体转染法将外源基因导入哺乳动物细胞,为此,我们对不同培养基配方进行摸索,确定了最佳的转染条件。更多还原

【Abstract】 Mammalian salivary glands belong to external secretion glands, but they have part function of endocrine glands. Most secretions can reach mouth by the salivary genal tube, and work in stomach when pH>4. While a fraction of secretions can reach directly into the bloodstream. There are lots of salivary secreted proteins, in which a protein may have multifunction and several proteins always share the same function. So mammalian salivary gland can be used as bioreactors.Parotid secretory protein (PSP) is expressed especially in the parotid gland, as it is antibiotics in function, many people have interested in it. To understand the expression of PSP and the relationship with α -Amylase (AMY), we research the experession of these two genes in different postnatal stages in mouse and pig by RT-PCR and Q-PCR (fluorescence quantitative Polymerase Chain React). Result shows, PSP expressed highly in almost postnatal stages in mouse and pig, and expression level changing with the time. Furthermore, the trend of PSP and AMY keeps the same in all the time, suggest they shares coregulation. And results from Northern and Western blot show the same as Q-PCR.We analysis the full amino acid sequence of pig PSP, then four potential peptides were found as antigenic determinants, their sequence and location at pig PSP were list as fellows: 22-37aa, 41-59aa, 180 -195aa, C-terminal. Among them, Peptide- I (22-37aa,) shown the best, then it was chosen as antigen to to conjugate with KLH, to develop antiserum in rabbit. A polyclonal antibody specific for pig PSP was generated. We research PSP expression in protein level; result shows PSP expression in higher level than that of GAPDH. And a new variant of PSP was found in mouse, which can reacted with PSP-antiserum.In the second part, we located the transcription start site by 5’RACE ,result shows, we got 39bp longer cDNA sequence than the reported one, and we correct the first code of PSP ORF to its -22bp upstream;And we located the transcription start site on -19bp of the new ORF. We predicted the core promoter of PSP, result shows, PSP promoter is untypical one, TATA box was not include, And eight Inrs were found near the transcription start site, it proved the result of 5’RACE, and fit the character of Inr.We located PSP promoter by instant transfection. Firstly ,we successfully rebuild plsmid pEGFP-N1, and set up a serious of cell transfection vectors including different length of 5’regulation region by PCR, clone and subclone.We take Acc-M cell as transfection subject, and take primary parotid epithelium cells as control. And I found suitable transfection condition, the best ratio of lipofectamine/DNA was 1/3 pmol: 2μ l using serum-free medium, and the optimal time to observe was 24 hours after transfection.更多还原