| γδT cell is an important subset in vertebrates,which plays an essential role on anti-tumours immunity and immunoregulation. Vγ9Vδ2 T cell dominates in human peripheral blood, and it has two distinct pathways for anti-tumor immunity. One is a pathway depending on the recognization of TCR to the antigens, and the other is a natural killer-like pathway. In this study, we tried to over co-express Vγ9 and Vδ2 fragments in prokaryotic cells, so as to further study the crystal structure and biological function ofγδT cell.Objective:This study was to find the co-expression level of Vγ9 and Vδ2 in different plasmids, to optimize the conditions to over-express Vγ9Vδ2, so as to explore the structure basis for Vγ9Vδ2 TCR to recognize the antigens and search for the unknown ligands, to provide a theoretical basis and research direction for utilizingγδT cell on clinical anti-tumour therapy and immuno- regulation.Method:1. Acquirement of the full gene sequences of Vγ9Vδ2 T cell receptor. Drew blood from volunteer's ulnar vein, anticoagulated and added PBS to isolate mononuclearcells by Ficoll gradient centrifugation. After isolated human peripheral mono-nuclear cells, we used total RNA extraction and RT-PCR to acquire and amplificate the gene of theγchain and theδchain on Vγ9Vδ2 T cells.2. Constructed the co-expression fragment of Vγ9 and Vδ2.Amplificated the Vγ9 and Vδ2 domain separately by specific primers that contained the linker gene, and integrated Vγ9 to Vδ2 by overlap PCR.3. Optimization of the vector condition to co-express Vγ9 and Vδ2. Ligated Vγ9Vδ2 to vector pGEX-6p-1,pET28a,pET32a and pET43.1a respectively. After expression at a small scale, we inspected the different expression level of Vγ9Vδ2 with different vectors.4. Over expression and purification of Vγ9Vδ2.Cultured the E coli that were transformed with Vγ9Vδ2-pET43.1a, which was better for expression, in a large medium.Then purifed the Vγ9Vδ2 protein by Ni-NTA after schizolysis.Results:1. The full correct nucleotide sequence ofγchain andδchain from Vγ9Vδ2 T cell.γδT cell has a very small proportionality in human peripheral blood,only about 5% in total T lymphocytes. Because the little abundance, the most investigators first stimulated peripheral T lymphocytes by inducer. Then amplificatedγδT cells and extracted target RNA fromγδT cell line.Although that ,we directly extracted RNA from mononuclearcells after isolated from peripheral blood,reverse transcripted and did gel extraction followed by ligated the gene fragment to cloning vector pEASY-T3.The sequencing result showed that they were full gene sequences of Vγ9Vδ2 T cell receptors absolutely.2. Integrated the Vγ9 and Vδ2 regions.Vγ9Vδ2 T cell receptor is a heterodimer,composed byγandδchains.Althoughγandδchains both contain V,(D,forδchain),J and C regions, it is their V region to recognize and interact with the antigens. In this study,we first amplificated theγchain's V region Vγ9 and theδchain's V region Vδ2 with specific primers,then ligated each other by linker(Gly4Ser)4,so as to co-express them.On one hand, it was convenient for expression.On the other hand, it was more natual for studying the structure and the function of Vγ9Vδ2 T cell receptor.3. Optimization of the vector condition. As a membrane protein, the poor expression level of Vγ9Vδ2 T cell receptor is a huge obstacle for exploring its structure and function. It does not express in many plasmids and strains, or express as inclusion body. However, we need it expressed in supernatant for study. According our experience and documents, contracted to classical plasmids pGEX-6p-1, pET28a and pET32a, we found that Vγ9Vδ2 expressed abundantly in pET43.1a, and located in supernatant. 4. Expression of Vγ9Vδ2-pET43.1a at a large scale and its purification. Transformed the recombinant vector Vγ9Vδ2-pET43.1a to the competent cells BL21 (DE3). After cultured in abundant medium, schizolysed the bacteria, centrifuged and gained the supernatant to flow through the column Ni-NTA to purify target protein. At this time, protein Vγ9Vδ2 was fused with the tag NusA from pET43.1a. It demonstrated that we had got the purified recombinant Vγ9Vδ2-NusA.5. Separation of Vγ9Vδ2 and NusA tag. Digested the recombinant protein Vγ9Vδ2-NusA by precission protease, and ensured the digested result with the specific antibody for Vδ2.Conclusion:By extracted total RNA from the isolated mononuclearcells in human peripheral blood, after reverse transcription we could acquire the whole gene of Vγ9Vδ2 T cell receptor's two chains. Not only that, the recombinant Vγ9Vδ2 co-expression fragment,ligated by linker, could well express in plasmid pET43.1a. And, it was easily separated from the NusA tag on the vector.
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