鲤肠道小肽转运载体PepT1多克隆抗体的制备及其组织表达分析

Preparation of the antibody and tissue distribution of the peptide transporter PepT1 in Cyprinus carpio L.

为从蛋白水平研究小肽转运载体（PepT1）在鲤（Cyprinus carpio L.）不同组织中的表达及分布规律,本研究采用PCR法获得PepT1 cDNA片段,并转化至大肠杆菌Rosetta,对目标多肽进行原核表达,将Pep T1重组蛋白纯化后免疫新西兰长耳兔（Oryctolagus cuniculus）,获取兔抗鲤PepT1多克隆抗体。采用ELISA检测抗体效价,免疫组化检测PepT1的组织表达情况,并用荧光实时定量PCR技术检测PepT1转录水平组织表达情况。结果显示,目标多肽分子量约为28 kD;抗体效价达到4×10~5。PepT1在鲤的前肠、中肠、后肠、脾、肝胰脏和肾中均有表达。肠道组织PepT1的高表达与其主要完成食物中肽的吸收功能密切相关,且吸收部位主要集中在前肠和中肠;肾中PepT1免疫染色阳性区域也较为明显,这与肾小管基底膜存在对短肽的重吸收功能相关。此外,肝胰脏和脾PepT1也有一定量的表达,可能与这两个重要器官代谢旺盛有关。本研究制备的兔抗鲤PepT1多克隆抗体能够有效识别鲤各组织中的PepT1蛋白,在后续研究中亦可用于其他鱼类PepT1转运蛋白的表达定位和定量研究。

The lack of a PepT1 antibody for fish has hindered analysis of PepT1 protein expression by immune tissue chemistry or western blot. We analyzed the expression and distribution of PepT1 in Cyprinus carpio L. at the transcriptional and protein levels. The immunogenic cDNA of PepT1 was obtained by PCR and the fragments were inserted into a pET-32a （＋） Vector and transformed into Escherichia coli Rosetta. The target polypeptide was expressed after induction with 1%o IPTG. The molecular weight of the recombinant protein was measured by SDS-PAGE electrophoresis. The purified PepT1 recombinant protein was used to immunize New Zealand long-eared rabbits by ear vein injection combined with subcutaneous injection for 38 d to obtain rabbit anti carp PepTl polyclonal antibody. Enzyme-Linked Immuno Sorbent Assay （ELISA） was used to evaluate the antibody titers, immunohistochemistry was used to check the tissue expression of PepT1, and real-time fluorescent quanti- tative PCR was used to evaluate the expression of PepT1 at the transcriptional level. The molecular weight of the target polypeptide was -28 kDa, and the antibody titer was 4× 105, suggesting that activity was high. The PepT1 protein was expressed in the foregut, midgut, hindgut, spleen, hepatopancreas, and kidneys. The level of expression was remarkably higher in the foregut and midgut than in other tissues, which may be due to their roles in absorption of peptides during digestion. The positive immune staining region in the renal tissue was obvious and clear, and consistent with short peptides being re-absorbed by PepT1 distributed on the renal tubular basement membrane. Additionally, the PepT 1 transporter was also expressed in the hepatopancreas and spleen, both metab- olically active tissues in carp. In conclusion, the rabbit anti-carp PepT1 polyclonal antibody prepared in this study can effectively identify PepT1 from different tissues of carp. The expression pattern of PepT1 is similar to that at the transcriptional level. Our results provide a foundation for study of the structure and function of PepT1 at both the molecular and protein levels in carp. Additionally, we provide a basis for analysis of the relationship between small peptide absorption and protein metabolism. The antibody described here will be an important tool for localization and quantitative research of PepT1 in related fish such as Carassius auratus, Ctenopharyngodon idellus, that belong to the family Cyprinidae or even in Cypriniformes.