目的提取、克隆BALB/c小鼠乳糖酶基因L ac,了解其序列及所编码蛋白的属性,并制备L ac cDAN探针,为进一步研究乳糖不耐受奠定基础。方法取4周龄BALB/c小鼠小肠组织,用T rizo l试剂盒提取总RNA,经RT-PCR、梯度PCR获取L ac cDNA。测序鉴定...目的提取、克隆BALB/c小鼠乳糖酶基因L ac,了解其序列及所编码蛋白的属性,并制备L ac cDAN探针,为进一步研究乳糖不耐受奠定基础。方法取4周龄BALB/c小鼠小肠组织,用T rizo l试剂盒提取总RNA,经RT-PCR、梯度PCR获取L ac cDNA。测序鉴定后将序列提交NCB I G enB ank,同时利用生物信息学,对其编码产物进行预测。探针的制备采用随机引物法以地高辛标记。结果所获得的L ac cDNA编码区有912 bp,编码303个氨基酸残基,在其二级结构中存在一个糖苷键水解酶基序,C-末端有一疏水螺旋区。标记后的探针经检测,灵敏度达到1 pg/μl。结论所获得的L ac基因经鉴定确为乳糖酶基因,由它编码的蛋白产物是乳糖酶。标记的探针灵敏度很高,可以用于原位杂交实验。展开更多
Background/Aims: Adult-type hypolactasia (primary lactose malabsorption) affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia ...Background/Aims: Adult-type hypolactasia (primary lactose malabsorption) affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia has been difficult to establish because of unsatisfactory diagnostic methods. C/T 13910 single nucleotide polymorphism residing 13910 base pairs from the 5′end of the lactase gene has been shown to be associated with lactase persistence. The aim of the study was to assess the applicability of the C/T-13910 variant as a diagnostic test for adult-type hypolactasia during childhood. Methods: Intestinal biopsies were obtained from 329 children and adolescents of African, Finnish, and other White origins aged 0.1-20 years undergoing upper gastrointestinal endoscopy because of abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activity and genotyped for the C/T-13910 variant using polymerase chain reaction minisequencing. Results: The frequency of the C/C-13910 genotype defining lactase non-persistence was well in agreement in this study with published figures for the prevalences of adult-type hypolactasia in Africans and Whites. The C/C-13910 genotype was associated with very low lactase activity (<10 U/g protein) in the majority of children tested at 8 years of age and in every child older than 12 years of age giving a specificity of 100% and sensitivity of 93% for the genetic test. The decline of lactase activity was somewhat earlier in African compared with Finnish children with C/C -13910 genotype (p < 0.03). Conclu sions: Genetic test of C/T -13910 polymorphism can be used as a first stage scr eening test for adult-type hypolactasia.展开更多
Objective. Adult lactose intolerance, which affects the majority of the population in the world, has been associated with a single nucleotide polymorphism, C- 13910T, located upstream of the lactase gene. Material and...Objective. Adult lactose intolerance, which affects the majority of the population in the world, has been associated with a single nucleotide polymorphism, C- 13910T, located upstream of the lactase gene. Material and methods. Adult patients undergoing lactose tolerance tests with lactose challenge and plasma glucose measurements were included in the study comprising 44 Swedes and 7 non- Swedish individuals. A real- time PCR method was established for the genotyping. Results. Out of 51 patients 48 had concordant results on genotyping and lactose tolerance tests, e.g. - 13910T/T and - 13910C/T genotypes had high glucose elevations. All patients with the heterozygous genotype, - 13910C/T, had high glucose elevations, and no gene- dose relationship was observed when comparing maximal glucose increases for cases with - 13910C/T and - 13910T/T genotypes. Conclusions. Genotyping could replace lactose challenge as a first- stage screening test in adults of European descent, but should be used together with tolerance tests in children and patients where secondary lactose intolerance is suspected.展开更多
文摘目的提取、克隆BALB/c小鼠乳糖酶基因L ac,了解其序列及所编码蛋白的属性,并制备L ac cDAN探针,为进一步研究乳糖不耐受奠定基础。方法取4周龄BALB/c小鼠小肠组织,用T rizo l试剂盒提取总RNA,经RT-PCR、梯度PCR获取L ac cDNA。测序鉴定后将序列提交NCB I G enB ank,同时利用生物信息学,对其编码产物进行预测。探针的制备采用随机引物法以地高辛标记。结果所获得的L ac cDNA编码区有912 bp,编码303个氨基酸残基,在其二级结构中存在一个糖苷键水解酶基序,C-末端有一疏水螺旋区。标记后的探针经检测,灵敏度达到1 pg/μl。结论所获得的L ac基因经鉴定确为乳糖酶基因,由它编码的蛋白产物是乳糖酶。标记的探针灵敏度很高,可以用于原位杂交实验。
文摘Background/Aims: Adult-type hypolactasia (primary lactose malabsorption) affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia has been difficult to establish because of unsatisfactory diagnostic methods. C/T 13910 single nucleotide polymorphism residing 13910 base pairs from the 5′end of the lactase gene has been shown to be associated with lactase persistence. The aim of the study was to assess the applicability of the C/T-13910 variant as a diagnostic test for adult-type hypolactasia during childhood. Methods: Intestinal biopsies were obtained from 329 children and adolescents of African, Finnish, and other White origins aged 0.1-20 years undergoing upper gastrointestinal endoscopy because of abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activity and genotyped for the C/T-13910 variant using polymerase chain reaction minisequencing. Results: The frequency of the C/C-13910 genotype defining lactase non-persistence was well in agreement in this study with published figures for the prevalences of adult-type hypolactasia in Africans and Whites. The C/C-13910 genotype was associated with very low lactase activity (<10 U/g protein) in the majority of children tested at 8 years of age and in every child older than 12 years of age giving a specificity of 100% and sensitivity of 93% for the genetic test. The decline of lactase activity was somewhat earlier in African compared with Finnish children with C/C -13910 genotype (p < 0.03). Conclu sions: Genetic test of C/T -13910 polymorphism can be used as a first stage scr eening test for adult-type hypolactasia.
文摘Objective. Adult lactose intolerance, which affects the majority of the population in the world, has been associated with a single nucleotide polymorphism, C- 13910T, located upstream of the lactase gene. Material and methods. Adult patients undergoing lactose tolerance tests with lactose challenge and plasma glucose measurements were included in the study comprising 44 Swedes and 7 non- Swedish individuals. A real- time PCR method was established for the genotyping. Results. Out of 51 patients 48 had concordant results on genotyping and lactose tolerance tests, e.g. - 13910T/T and - 13910C/T genotypes had high glucose elevations. All patients with the heterozygous genotype, - 13910C/T, had high glucose elevations, and no gene- dose relationship was observed when comparing maximal glucose increases for cases with - 13910C/T and - 13910T/T genotypes. Conclusions. Genotyping could replace lactose challenge as a first- stage screening test in adults of European descent, but should be used together with tolerance tests in children and patients where secondary lactose intolerance is suspected.
