Cloning Of HSA-TP5 Fusion Protein CDNA And It Expression In Pichia Pastoris

Thymopentin(TP5) is pentapeptide corresponding to the amino acids 32-36of thymopoietin and exhibiting the full biological activity of the naturalhormone. It is an immunomodulator which displays two-ways immuneregulation function, induces the cytodifferentiation of T cells and promotes thedevelopment and activation of T subgroup. It has many functions such asenhancing the phagocytosis of rhagiocrine cells, reinforcing immune function ofrhodocyte, strengthing cytoactive of natural killer cell, increasing productionlevel of IL-2 and expression level of IL-2 receptor, strengthening SOD enzymeactivity in serum. Thymopentin has been widely used in clinic, which achievesgood therapeutic efficacy in malignant tumor, chronic hepatitis, infectiousdiseases,and so on. However, thymopentin we used is almost production ofsolid-phase synthesis, and reports on gene engineering production ofthymopentin have not been seen domestically.Human serum albumins (HSA) generally distribute the exterior and interiorof blood vessel, and are main components of human serum, which are chargedwith the task of maintaining normal osmotic pressure of blood and transportinghydrophilia molecules;clinically, HSA, a kind of material for fluid expansion iswidely used in hemorrhoea, shock, empyrosis, cancer, leukocytosis,hypoalbuminosis and so on. HSA is stable in body, long in half life (19d), absentof enzymic and immunological function and a natural carrier for variousmolecules and drug. It has been studied for a fusion carrier of drugs in manylaboratories. Thymopentin is unstable in body, short in half-life (only 30s), fastin degradation. In order to prolong half-life of thymopentin and reinforce itsstability, we established the HSA-TP5 fusion carrier, and finished its expressionand identification in Pichia pastoris. Detailed jobs were showed as below:1 Construction of HSA Cloning VectorOn the basis of HSAcDNA sequence published in genebank, we designedprimers, extrcted total RNA from fetal liver tissues, obtained HSA cDNA byRT-PCR, cloned HSA cDNA into pMD18-T vector, and establishedrecombinant cloning vector pMD18-T-HSA with signal peptide sequence andtotal encoding sequence of HSA. The designed sequence identified withpublished sequence by enzyme digesting and DNA sequencing, structuring abase for establishing HSA-TP5 expression vector later.2 construction of HSA-TP5 expression vectorAccording to the principle of primer design, we used cloning vector withcorrect sequence as a template to design expression primers: inserting enzymecutting sites, BstBI and KpnI, and HSA signal peptide sequence into forwardprimer and reverse primer, and inducing pentapeptide sequence before5'terminator codon of reverse primer. We amplified encoding sequence ofHSA-TP5 fusion gene by PCR, cloned it into pPICZαexpression vector. DNAsequencing verified that we obtained correct pPICZα-HSA-TP5 recombinationexpression vector.3 Expression and Identification of HSA-TP5 in Pichia pastorisWe electrotransformated linearrizated plasmid pPICZα-HSA-TP5 intocompetent cell, Pichia pastoris X-33, then smear the cells on YPD agar platecontaining Zeocin, and cultured them 48h-72h at 28 ℃. We choosed 9 cloneswith Zeocin-resistance to carry on verification. In the process we used thetemplate of genome DNA and expression primers.We amplified positive converter with shake cultivation at 28℃ , inducedthem to express protein with 0.5% methyl alcohol, and then identifiedsupernatant protein with SDS-PAGE analysis. We purified HSA-TP5 fusionprotein with cation-exchange chromatography and hydrophobicchromatography and test its cytoactive with MTT method. Resultsdemonstrated: in SDS-PAGE analysis obtaining a protein of 68kD molecularweight, representing interest protein;in cytoactive verification confirming thatHSA-TP5 can stimulate proliferation and differentiation of T lymphocyte.To sum up: in the present experiment we firstly constructed TP5-HSAfusion gene by the technology of genetic recombination, and successfullyobtained expression product in Pichia pastoris ,a eukaryotic expression system,and observed that HSA-TP5 fusion protein stimulated proliferation anddifferentiation of lymphocyte, demonstrating that infusion reaction betweenthymopentin and HAS did not destroy biologic activity of thymopenti. Ourresearch was a successful application of HSA infusion technology and exploiteda new technology method to product thymopentin, and supplied theory base forfurther developing other small peptide sequence.