| The placenta, providing the nutrients for mammalian viviparous animals during their time of pregnancy, is the organ for the embryo growth. Its tissue cell includes the material ingredients needed for the embryo growth, such as the rich steroid hormone, the peptide hormone, the prostaglandin, the growth factor, embryos cell factor and so on. Since Professor Shan Dao of Japanese Kyoto hospital successfully extracted hormone from the goat placenta in 1943, plenty of scholars have been interested in studying the extraction from goat placenta. Recent years studies have discovered that the goat placental extract contains various bioactive substances, including the small molecules of peptide. However, the reports on the purification and researches of the components of the sheep placental extract functional components are hard to find. In this study, the researcher made the goat placenta extraction by means of tissue mashing, repeated freezing and thawing, refrigerated centrifuge and ultrafiltration. Then I used the Tricine-SDS-PAGE electrophoresis to identify the presence of placental peptide. By RP-HPLC separation step by step, I purified liquid peptide components in the goat placenta extract and detected immunity activity of the peptide fraction by using E-rosette test. To get the amino acid sequence of primary structure, by the method of Edaman degradation, I measure the N-terminal amino acid sequence of active components. The purpose is to purify placenta peptide with immune activity by ultrafiltration and RP-HPLC and to study the purified peptide immune activity and identify the N-terminal amino acid sequence which supply necessary evidence for clarifing its biological activity mechanism and further study the small molecule with immunity activity.The study as follows:1, goat placenta extract were prepared by tissue mashing, freezing and thawing combining with ultrafiltration; 2, the goat placenta extract of physical and chemical properties were identified, and identified the peptide existence of the goat placenta by Tricine-SDS-PAGE electrophoresis; 3, the prepared goat placenta were purified one by one until obtains the purity quite high sole component by using the RP-HPLC method, and the immunity active of each purification was detected by using the E-rosette test; 4, the high activity of immune and high purity component N-terminal amino acid sequence were determined by Edman degradation.The results as follows:firstly, the goat placenta extract is colorless or slightly yellow transparent liquid, and is porous sponge-like white powder after being freeze-drying. Besides, its PH is 6.40±0.02. And the reaction of protein characterization indicates that the color has become purple. The reaction of protein precipitation in response is negative. The electrophoresis result has showed that the goat placenta extract includes the small molecular peptide class material and its peptide content is 2.14±0.05mg/ml. It has two absorption peaks between 190nm and 340nm. one is near 212nm and the other is near 253nm. secondly, RP-HPLC has purified out the three components of high purity. They are peak 1-5, peak 1-7 and peak 2-3. Its purity quotient has reached, respectively,90.6% purity,91.4%,99.6%. Thirdly, by E-rosette test, the result of the activity detection of the three components with high purity has showed that peaks 1-5 and peaks 2-3 have immunity activity.The N-terminal first 10 amino acid sequence of peak 2-3 is Trp-Glu-Pro-Asn-Val-Ser-Ala-His-Ala-Phe by the method of Edman degradation. And logging this sequence in the U.S. National center for biotechnology information (NCBI) and applying of the short sequence on the BLAST search tools of protein database search match, I find no homology with this peptide sequence. So it is a new peptide. However, the stability of the peptide immunity and its mechanism of action still need further study.Conclusion:(1) The goat placenta extract with its pH6.40±0.02 and a polypeptide content of 2.14±0.05mg/ml has been acquired by repeated freeze-thaw method and ultrafiltration.(2) Three peptide components, the peak 1-5, the peak 1-7 and the peak 2-3 have been purified by using the RP-HPLC gradually separating and purifying the goat placenta extract and the purity quotient of the three, respectively, is 90.6%,91.4%,99.6%. The E-rosette test has discovered that peak 1-5, peak 2-3 components can strengthen the function of the dereceptor lymphocyte superficial receptor recovery, and can promote human beings circumference blood T lymphocyte E-rosette formation, and also has the dose effect dependence which have indicated that it has the T cellular immunity activity.(3) The Edman degeneration has determined amino acid sequence of peak 2-3 and has discovered the first ten amino acid sequence of the N-terminal is Trp-Glu-Pro-Asn-Val-Ser-Ala-His-Ala-Phe. After the Blast protein database retrieval, this peptide has been discovered as a new peptide.
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