关于TIGD7课题

TIGD7蛋白是一个在高等哺乳动物中极度保守的蛋白，从人的蛋白序列保守性上看，可认为是在灵长类，甚而可局限在人这一物种中所特有的蛋白。

TIGD7基因

TIGD7基因序列及蛋白序列

NCBI Reference Sequence: NM_033208.4，

       1 M  N  K  R  G  K  Y  T  T  L  N  L  E  E  K  M  K  V  L  S  
       1 ATGAATAAGAGAGGGAAATATACAACACTGAATTTGGAGGAGAAAATGAAGGTTCTAAGT
      21 R  I  E  A  G  R  S  L  K  S  V  M  D  E  F  G  I  S  K  S  
      61 AGAATTGAAGCTGGACGATCACTTAAAAGTGTAATGGATGAATTTGGAATCAGTAAGTCA
      41 T  F  Y  D  I  K  K  N  K  K  L  I  L  D  F  V  L  K  Q  D  
     121 ACATTTTATGACATTAAAAAAAATAAGAAGTTAATTCTGGACTTTGTACTGAAGCAGGAC
      61 M  P  L  V  G  A  E  K  R  K  R  T  T  G  A  K  Y  G  D  V  
     181 ATGCCATTAGTAGGGGCTGAGAAGAGAAAGAGGACAACGGGAGCCAAATATGGTGATGTA
      81 D  D  A  V  Y  M  W  Y  Q  Q  K  R  S  A  G  V  P  V  R  G  
     241 GATGATGCGGTCTACATGTGGTACCAACAGAAACGCTCAGCCGGTGTTCCGGTAAGAGGC
     101 V  E  L  Q  A  A  A  E  R  F  A  R  C  F  G  R  T  D  F  K  
     301 GTGGAGCTTCAGGCTGCTGCAGAGAGATTTGCACGGTGTTTTGGGCGAACAGATTTCAAA
     121 A  S  T  G  W  L  F  R  F  R  N  R  H  A  I  G  N  R  K  G  
     361 GCTAGCACTGGTTGGCTTTTTAGATTTCGAAATCGGCATGCAATTGGGAACCGAAAAGGA
     141 C  G  E  Q  V  L  S  S  V  S  E  N  V  E  P  F  R  Q  K  L  
     421 TGTGGGGAACAAGTCCTAAGTTCAGTTTCTGAAAATGTTGAGCCATTTCGACAAAAACTG
     161 S  M  I  I  K  E  E  K  L  C  L  A  Q  L  Y  S  G  D  E  T  
     481 TCCATGATAATCAAAGAGGAGAAACTGTGTCTAGCTCAGCTATACAGTGGGGATGAAACA
     181 D  L  F  W  K  S  M  P  E  N  S  Q  A  S  R  K  D  I  C  L  
     541 GACCTCTTTTGGAAGTCAATGCCAGAAAATTCTCAGGCAAGTAGGAAAGATATCTGCCTA
     201 P  G  K  K  I  N  K  E  R  L  S  A  F  L  C  A  N  A  D  G  
     601 CCAGGGAAGAAAATAAACAAAGAAAGGTTGTCTGCCTTTTTATGTGCAAATGCAGACGGA
     221 T  H  K  L  K  S  I  I  I  G  K  S  K  L  P  K  S  V  K  E  
     661 ACTCATAAATTAAAGTCAATCATTATTGGAAAATCAAAACTGCCCAAAAGTGTGAAAGAG
     241 D  T  S  T  L  P  V  I  Y  K  P  S  K  D  V  W  F  T  R  E  
     721 GACACAAGTACATTGCCTGTGATATATAAACCCAGTAAAGATGTTTGGTTCACCAGAGAA
     261 L  F  S  E  W  F  F  Q  N  F  V  P  E  V  R  H  F  Q  L  N  
     781 TTGTTTTCAGAATGGTTTTTTCAAAACTTTGTTCCTGAGGTCCGACATTTTCAACTTAAT
     281 V  L  R  F  H  D  E  D  V  R  A  L  L  L  L  D  S  C  P  A  
     841 GTTCTAAGATTTCATGACGAGGACGTCAGGGCATTGTTACTTCTGGACAGTTGCCCGGCT
     301 H  P  S  S  E  S  L  T  S  E  D  G  R  I  K  C  M  F  F  P  
     901 CATCCTTCCTCTGAATCCCTAACCAGTGAGGATGGTCGAATAAAATGTATGTTCTTCCCC
     321 H  N  T  S  T  L  I  Q  P  M  N  Q  G  V  I  L  S  C  K  R  
     961 CATAACACTTCAACCTTGATTCAACCAATGAATCAAGGTGTGATCTTGAGCTGCAAACGG
     341 L  Y  R  W  K  Q  L  E  E  S  L  V  I  F  E  E  S  D  D  E  
    1021 CTGTATAGATGGAAGCAACTTGAAGAGAGTCTTGTAATATTTGAAGAAAGTGATGATGAG
     361 Q  E  K  G  D  K  G  V  S  K  I  K  I  Y  N  I  K  S  A  I  
    1081 CAAGAGAAAGGAGATAAAGGAGTTTCCAAAATTAAAATCTACAATATAAAAAGTGCAATT
     381 F  N  W  A  K  S  W  E  E  V  K  Q  I  T  I  A  N  A  W  E  
    1141 TTTAACTGGGCAAAAAGTTGGGAAGAAGTAAAACAAATAACCATAGCAAATGCATGGGAA
     401 N  L  L  Y  K  K  E  P  E  Y  D  F  Q  G  L  E  H  G  D  Y  
    1201 AATCTTCTTTACAAAAAGGAACCTGAATATGATTTTCAAGGCTTAGAACATGGGGATTAT
     421 R  E  I  L  E  K  C  G  E  L  E  T  K  L  D  D  D  R  V  W  
    1261 AGAGAAATTCTTGAAAAATGTGGGGAGTTGGAAACTAAGTTGGATGATGATAGGGTGTGG
     441 L  N  G  D  E  E  K  G  C  L  L  K  T  K  G  G  I  T  K  E  
    1321 TTAAATGGAGATGAAGAAAAGGGTTGTCTCCTAAAAACCAAAGGTGGAATAACAAAGGAA
     461 V  V  Q  K  G  G  E  A  E  K  Q  T  A  E  F  K  L  S  A  V  
    1381 GTTGTTCAAAAAGGAGGAGAAGCTGAGAAGCAGACTGCTGAATTTAAATTATCTGCTGTA
     481 R  E  S  L  D  Y  L  L  D  F  V  D  A  T  P  E  F  Q  R  F  
    1441 AGAGAGAGTTTGGACTACCTTCTTGACTTTGTTGATGCCACACCTGAGTTTCAGAGATTC
     501 H  F  T  L  K  E  M  Q  Q  E  I  V  K  K  Q  F  Q  S  K  I  
    1501 CACTTCACACTGAAAGAGATGCAACAAGAAATAGTCAAGAAACAGTTCCAGAGTAAAATC
     521 H  S  R  I  G  S  F  L  K  P  R  P  H  N  I  K  D  S  F  S  
    1561 CATTCTAGAATTGGTAGCTTTTTGAAACCTAGGCCTCATAATATTAAGGACTCCTTCAGT
     541 G  P  S  T  S  G  S  N  H  *  
    1621 GGGCCTTCAACTTCTGGTTCTAATCATTAG

患者表现及突变

• 临床表现：父母近亲结婚，患者均幼年发病，双上肢抖动（弟弟为姿势性震颤，姐姐兼有静止性及姿势性震颤），共济失调，智能发育较差(也可能是认知障碍)，皮肤色素异常沉着，锥体束征阳性
• 肌电图：多发性感觉运动性周围神经病，以脱髓鞘病变为主
• MRI:大脑萎缩，脑室扩大，脑白质病变
• cDNA突变：c.823C>T，p.R275X，截断突变

       1 M  N  K  R  G  K  Y  T  T  L  N  L  E  E  K  M  K  V  L  S  
       1 ATGAATAAGAGAGGGAAATATACAACACTGAATTTGGAGGAGAAAATGAAGGTTCTAAGT
      21 R  I  E  A  G  R  S  L  K  S  V  M  D  E  F  G  I  S  K  S  
      61 AGAATTGAAGCTGGACGATCACTTAAAAGTGTAATGGATGAATTTGGAATCAGTAAGTCA
      41 T  F  Y  D  I  K  K  N  K  K  L  I  L  D  F  V  L  K  Q  D  
     121 ACATTTTATGACATTAAAAAAAATAAGAAGTTAATTCTGGACTTTGTACTGAAGCAGGAC
      61 M  P  L  V  G  A  E  K  R  K  R  T  T  G  A  K  Y  G  D  V  
     181 ATGCCATTAGTAGGGGCTGAGAAGAGAAAGAGGACAACGGGAGCCAAATATGGTGATGTA
      81 D  D  A  V  Y  M  W  Y  Q  Q  K  R  S  A  G  V  P  V  R  G  
     241 GATGATGCGGTCTACATGTGGTACCAACAGAAACGCTCAGCCGGTGTTCCGGTAAGAGGC
     101 V  E  L  Q  A  A  A  E  R  F  A  R  C  F  G  R  T  D  F  K  
     301 GTGGAGCTTCAGGCTGCTGCAGAGAGATTTGCACGGTGTTTTGGGCGAACAGATTTCAAA
     121 A  S  T  G  W  L  F  R  F  R  N  R  H  A  I  G  N  R  K  G  
     361 GCTAGCACTGGTTGGCTTTTTAGATTTCGAAATCGGCATGCAATTGGGAACCGAAAAGGA
     141 C  G  E  Q  V  L  S  S  V  S  E  N  V  E  P  F  R  Q  K  L  
     421 TGTGGGGAACAAGTCCTAAGTTCAGTTTCTGAAAATGTTGAGCCATTTCGACAAAAACTG
     161 S  M  I  I  K  E  E  K  L  C  L  A  Q  L  Y  S  G  D  E  T  
     481 TCCATGATAATCAAAGAGGAGAAACTGTGTCTAGCTCAGCTATACAGTGGGGATGAAACA
     181 D  L  F  W  K  S  M  P  E  N  S  Q  A  S  R  K  D  I  C  L  
     541 GACCTCTTTTGGAAGTCAATGCCAGAAAATTCTCAGGCAAGTAGGAAAGATATCTGCCTA
     201 P  G  K  K  I  N  K  E  R  L  S  A  F  L  C  A  N  A  D  G  
     601 CCAGGGAAGAAAATAAACAAAGAAAGGTTGTCTGCCTTTTTATGTGCAAATGCAGACGGA
     221 T  H  K  L  K  S  I  I  I  G  K  S  K  L  P  K  S  V  K  E  
     661 ACTCATAAATTAAAGTCAATCATTATTGGAAAATCAAAACTGCCCAAAAGTGTGAAAGAG
     241 D  T  S  T  L  P  V  I  Y  K  P  S  K  D  V  W  F  T  R  E  
     721 GACACAAGTACATTGCCTGTGATATATAAACCCAGTAAAGATGTTTGGTTCACCAGAGAA
     261 L  F  S  E  W  F  F  Q  N  F  V  P  E  V  *  
     781 TTGTTTTCAGAATGGTTTTTTCAAAACTTTGTTCCTGAGGTCTGA

用到的引物

• 为了验证突变蛋白的表达情况（表达量、表达部位），使用 pIRES2-EGFP载体，带Flag标签，以GFP作为表达量内参。设计引物：

• 为了在果蝇中研究突变蛋白的功能，使用果蝇基因表达载体pUAST载体，构建with / without Flag标签的插入序列，设计引物：
• EcoR I, Xho I双酶切buffer： 1 x H

• 为了在斑马鱼中研究突变蛋白的功能，使用体外转录载体pCS2+载体，带Flag标签，设计引物：