Human salivary cystatin S. Cloning, sequence analysis, hybridization in situ and immunocytochemistry.
Human salivary cystatin S. Cloning, sequence analysis, hybridization in situ and immunocytochemistry.
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人唾液胱抑素 S。克隆、序列分析、原位杂交和免疫细胞化学。
DOI：
10.1042/bj2780627
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Levine,MJ
中科院分区：
文献类型：
--
作者：
Bobek,LA;Aguirre,A;Levine,MJ
在λ gt 11 Sfi-Not定向特异性表达载体中构建人下颌下腺（SMG）cDNA文库，然后用产生的抗人唾液半胱氨酸蛋白酶抑制剂的抗体筛选。克隆C4-4编码N-末端截短的半胱氨酸蛋白酶抑制剂S，而其它克隆编码半胱氨酸蛋白酶抑制剂SN。然后用C4-4重新筛选文库，并通过线性PCR直接从洗脱的噬斑中扩增若干阳性克隆的插入片段，并通过Southern印迹和直接DNA测序分析PCR产物。C3和C12两个克隆编码全长的分泌型胱抑素S及其前导肽，包括5′和3′非翻译区。这些克隆与编码人唾液半胱氨酸蛋白酶抑制剂SN的cDNA克隆和编码半胱氨酸蛋白酶抑制剂SN和SA的基因组克隆显示出高度的序列相似性。正常人SMG和腮腺（PG）组织切片原位杂交定位的胱抑素基因转录的浆液腺泡细胞的两个腺体的细胞质中，具有更高浓度的胱抑素mRNA的SMG。免疫细胞化学定位的唾液胱抑素基因产物也浆液细胞，和胱抑素蛋白的水平与胱抑素mRNA的量相关，与一个更强的信号在SMG比PG。
A human submandibular-gland (SMG) cDNA library was constructed in a lambda was constructed in a lambda gt11 Sfi-Not orientation-specific expression vector and then screened with antibody generated against human salivary cystatins. The clone C4-4 encoded an N-terminally truncated cystatin S, whereas the others encoded cystatin SN. The library was then rescreened with the C4-4, and the inserts of several positive clones were directly amplified from the eluted plaques by linear PCR and the PCR products analysed by Southern blotting and direct DNA sequencing. Two clones (C3 and C12) encoded a full-length secreted cystatin S and its leader peptide and included 5′- and 3′-untranslated regions. These clones showed a high degree of sequence similarity to cDNA clones encoding human salivary cystatin SN and genomic clones encoding cystatin SN and SA. Hybridization in situ of normal human SMG and parotid-gland (PG) tissue sections localized the cystatin-gene transcripts to the cytoplasm of serous acinar cells of both glands, with a much higher concentration of cystatin mRNA in the SMG. Immunocytochemistry localized the salivary cystatin gene products also to the serous cells, and the levels of cystatin protein correlated with the amount of cystatin mRNA, with a much stronger signal in the SMG than in the PG.