| The post-weaning decline of the intestinal lactase activity is a very common condition in many human ethnics all over the world, which is know as adult-type hypolactasia. There are many hazards to health along with hypolactasia, including malabsorption of calcium, potassium, and magnesium in dairy products, etc. At present, the mechanisms of latose intolerance(LI) are unknown, although many researches have been put in practice. The post-weaning decline of the intestinal lactase activity seen in human condition also occurs in other mammals such as pig, rat and rabbit, making them as animal models in m any s tudies. In o rder t o c larify t he e ffect o f 1 ong t erm d linking milk on lactase and the possible molecular mechanisms of LI by multi-generation breed trial, we choose the BALB/c inbred strain mouse as experimental animal, which has numerous advantages including its economically controlled genetic background and sequenced genome. Its reletive genetic information have been obtained from this study.The present study includes the following four parts:1 Molecular cloning and sequencing of a novel cDNA (Lac) from BALB/c mouse1.1 Method1.1.1 Cloning of Lac from BALB/c miceTotal RNA was extracted from intestinal tissue of 4-week BALB/c mouse. Specific RT-PCR primers were desinged according to human and rat lactase gene using software Primer5.0. The first chain of Lac cDNA was obtained from reverse transcription and Lac cDNA was amplified by gradient polymerase chain reaction.1.1.2 Construction of the recombinant plasmid pNEB-lacPCR fragment and cloning vector pNEB193 were degisted with the restriction endonucleases EcoR I and Sal I , then recombined with DNA ligase. The recombinant plasmids were transformed into competent E. coli TB1 cells. After screening and identification, the small-scale preparation of recombinant plasmid was sent to TaKaRa company for DNA sequencing. The data of Lac from BALB/c mice was directely submitted to NCBI GenBank. 1.1.4 Analysis of BALB/c mouse LacBioinformatics tools such as NCBI BLAST and Omiga2.0 were used to study the structure characteristics of Lac from BALB/c mouse and some of the functional features of its encoding product were predicted by these solftwares.1.2 Result1.2.1 BALB/c mice Lac edcoding region cloned in this study providedto be 912bp, start code is ATG, edcoding 303 amino acids.1.2.2 Lac sequence has been published on NCBI GenBank, Accession No.AY751548.1.2.3 The chemical characters of protein edcoded by Lac are as following:Protein: Lactase; Length: 303aa; MW: 34.113Kda; pI: 8.676; etc.1.2.4 There is a motif of Glycosyl hydrolase F1 in the secondary structure, which is located between 121-129aa with a sequence of IYVTENGVS.1.2.5. A transmembrane region has been found at the C-terminal of the peptide, which indicate that the structure is similar to that of human. 1.2.6 The alignment of BALB/c mice Lac with C57BL/6J mice, rat Lct, and human Lct is 99%, 88-89%, 83%, respectively. 1.3 Conclusion1.3.1 A novel lactase cDNA Lac has been cloned from BALB/c mouse.1.3.2 The recombinant plasmid pNEB-lac earring murine lactase encoding sequence Lac has been constructed.1.3.3 There are alignment sequences of BALB/c mouse Lac in human and rat genomes.2 Labeling of Lac probe and the study of determination method of lactase activity.1.1 Method1.1.1 The template of cDNA probe is a 318bp preservedfragment from the Lac cDNA h ydrolyzed by BamH I in Parti.1.1.2 Lac cDNA probe labeling was carried out w ith Dig HighPrime DNA Labeling and Detection Starter Kit 1 from Roche of Germany.1.1.3 The sensitive detection of probe was carried out with Sensitive Detection by in situ Kit from Boster in Wuhan.1.1.4 The determination method of lactase was the modification from Dahlqvist method.1.2 Result1.2.1 The sensitivity of cDNA probe is about 0.1pg1.2.2 The suitable pH is 4.6(mice), 5.6(rat).1.2.3 The best reaction condition is: water bath lasted for 75 min,developed color for 45min, 3.0ml of coloring reagent(TGO) was need.1.3 Conclusion1.3.1 The probe labeled in this study is sensitive.1.3.2 The method has a better repetability and precision.3 The effect of Long-term drinking milk on lactase of BALB/c mouse.3.1 AimsTo clarify the effect of long—term drinking milk on lactase of mice, and the potential mechanisms of LI.3.2 MethodBe carried out following the Part 2. mRNA was determined byin situ hybridization.3.3 Result3.3.1 There were differences of lactase activities between mice with different ages.3.3.2 Generation 1 was the same as the Generation 0, with a decline of lactase activity step by step.3.3.3 The lactase activities of the 7-week-old BALB/c mice in Generation 2 were similar to those of the 3-week-old mice.3.3.4 There were no significant differences of mRNA levels among mice in 3 generations.3.4 Conclusion3.4.1 Continuously intaking milk would probably enhance the lactase activity of mice.3.4.2 The regulation of lactase gene was carried out in the process of translationalor post-translational levels, but in the transcriptional level.4 The study on the lactase distribution along the small intestine of SD rats4.1 Method3-4wk-old SD rats were used for the study. From the duodenum, we scissored the intestine 3 parts continuously with about 10 cm long as the specimen.4.2 Results4.2.1 The lactase activities of the 3 parts were declined from the duodenum stage by stage.4.2.2 There were no significant differences of the mRNA levels among mice in 3 generations.CONCLUSIONS: The present study was the first report on the study of the effect of long-term drinking milk on lactase using BALB/c mice inbred strain as experimental animal model.We successfully isolated the total RNA of mice, and carried out the molecular cloning and sequencing of Lac cDNA. The information from the present study would provide a new approach for the comprehensively understanding of LI mechanisms. The results suggested that long-term drinking milk would maintain the lactase activity. The regulation of lactase gene might occur in the stage of translational and/or post-translational level, but the transcriptional level.
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