| Pig liver esterase(PLE)belongs to the serine hydrolase, it is a enzyme family which is comprised by a variety of isoenzymes whose gene sequence are highly homologous. PLE often show high stereoselectivities and activity when they catalyze the hydrolysis of substrates. So the PLE isoenzymes become the main role of hydrolytic enzymes in the field of organic synthesis and people rarely researched its role of drug metabolic enzyme. Previous study of our research group had suggested that most of sulfonamides can be hydrolyzed by PLE1, which is one of the main PLE isoenzymes. And the enzymatic activity of PLE isozymes exist differences. Isozyme abundance is possibly different in different pig breeds. Inspired by the high hydrolytic activity of PLE, and the human carboxylesterases expression level presents signally age-related correlation and has been proven to be the basis of clinical medication, and also based on the previous study of our research group, we systematically research the difference of PLE isozymes abundance and the total expression quantity in different breeds and ages of pig, it hoped to provide necessary theoretical basis for pigs’ clinical rational drug use.For the research of strain difference we choosed Tong cheng swine, which is national key protected species in China, and large white swine, which is the fine varieties abroad. For the research of PLE expression profile, according to the age group of human carboxylesterases expression profile, we choosed 8 days, 1month, 3months and adult(6-7months) this 4 age groups and each group including 3 pigs and they are unrelated individuals in 3 generations. Because of high homology of the gene and amino acid sequence between PLE isozymes, by means of RT-q PCR and WB we can’t distinguish the m RNA and protein expression level of each PLE. So we firstly designed PLE universal primers and prepared PLE universal antibodies which are based on the conservative nucleotide sequence and amino acid sequence of PLE isozymes. And they were used to detect the total expression levels of m RNA and protein. At the same time, we detected the hydrolytic activity of pig liver S9 samples towards p-NPA. Then we achieved the emerging frequency of each PLE isozyme by plenty of cloning, sequencing of PLE isozymic ORF. Combineing the total expression levels of PLE isozymes’ m RNA and the emerging frequency of each PLE isozyme obtained the expressive abundance data of each PLE isozyme.The main contents and results of the research by follows:Firstly, we detected the m RNA expression level of PLE isozymes of different organs(liver, heart, spleen, kidney, lung, small intestine, skin, muscle, brain, lymphaden and thymus) of 1month and adult of two breeds of swines though RT-q PCR. The results showed that the highest expression level happened in liver, then kidney, small intestine and skin in all the pigs. Liver is the major metabolic and detoxification organ of organism, and there is the highest expression level of PLE in liver, so in follow-up studies we concentrated our attentions on the liver’ PLE.We detected the total expression levels of PLE isozymes in the liver of two breeds of swines at four ages by RT-q PCR. The results turned out to be that the highest expression groups are 3months(Tong Cheng swine) and 1month(large white swine), respectively. The overall is upward trend. Except for 3 months group, The PLE m RNA expression levels of large white swine are higher than Tongcheng swine in the other 3 groups.We designed 2 polypeptide chains according to the conserved sequence of the end of the amino acid sequence, they are PLE-C1-1: SKEAAKKPPKIKC and PLE-C2: CNTQAAKRLKGEE. The 2 polypeptide chains were balanced mix and connected with KLH to formed immunogen. We used the immunogen to immune New Zealand rabbits, obtained immune serum. The immune serum was purified by immunoaffinity chromatography. Then we got high-titre and high specificity PLE universal antibodies. S9 samples prepared from the same age S9 with mixing the same amount of 3 pigs. We detected the total protein expression levels of PLE isozymes of S9 samples by WB. We got the gray values of PLE and GAPDH by Image J, and calculated the specific values of PLE and GAPDH, the results was that the specific values of 8days, 1month, 3months and adult pigs were 0.74,2.00,3.62,3.09(Tong cheng swine) and 1.30,3.86,2.89,3.82(large white swine) respectively. Except for 3 months group, The PLE protein expression levels of large white swine are higher than Tongcheng swine in the other 3 groups. The data showed that the protein expression levels of PLE enzymes were in accord with the expression levels of m RNA.We detected hydrolytic activity of liver S9 samples above in containing 1μmol/L p-NPA of PBS buffer solution(50m M, p H7.2) reaction system. And the liver S9 specific activity of 8 days, 1month, 3months and adult pigs are 0.19 U,0.59 U,0.9 U,1.29 U(Tong cheng swine) and 0.4 U,1.05 U,1.22 U,1.69 U(large white swine), respectively. The hydrolytic activity of S9 along with the age growth, it in sum agreed with the PLE m RNA and protein expression levels. The hydrolytic activity of S9 of large white swine were higher than that of Tong cheng swine, by and large, this is consistant with that PLE expression levels of large white were higher than Tong cheng swine at the same age.It’s still unknown the expression abundance of each PLE isozyme. We cloned the complete ORF of all subtypes of isoenzymes in each pig’s liver. At least randomly screen out 20 positive clones of bacteria for each pig and then sequenced. A total of more than 800 positive clones were sequenced. Analyzing the cloning results, we cloned 108 PLE subtypes in total, and there were 103 new subtypes except for 5 subtypes which had been reported. There were more than 50 subtypes which were repeated emergence, and 39 subtypes repeated more than 2 pigs, 19 subtypes were shared by two breeds of swines. Based on the similarity of amino acid types of the 25 variation site, we classified 54 subtypes which had high frequency to 7 classes(A-G) and each major category was distinguished by arabic numerals.According to the frequency of PLE isozyme appeared in every age stage, we found there are obvious differences in PLE expressive abundance between two breeds of swines. PLE-A1 was the highest expressed and PLE-B9 was second expressed in 4 age stages about Large White swine, on the contrary, PLE-B9 were the highest expressed and PLE-A1(8days), PLE-G5(1month and 3months) were the second expressed about Tong cheng swine. PLE-G3 was the highest expressed in adult Tong cheng swine. There were a sharp rise of expression of PLE-G5 and PLE-G3 in growth periods and mature periods(6-7months), it prompted that PLE-G5 and PLE-G3 may play important roles in growth periods and mature periods respectively.This study found significant varietal difference in PLE expression in liver of Tong Cheng swine and large White swine, and its expression level was significantly associated with age, The difference of expression profile and varieties of Tong Cheng swine and Large White swine’s PLE in liver will lead to the differences of the efficacy and toxicity to the same veterinary drugs for different ages and different pig breeds. Therefore, the use of related veterinary drugs should be adjusted according to the pig’s variety and age. Data obtained from this research and the subsequent research results can provide the necessary theoretical basis for the rational use of pig clinical medicine, which are veterinary drugs’ hydrolysis characteristics using a large number of new PLE isozymes obtained in this study. |
PDF Full Text Request