Study On Cloning Of A Translationally Controlled Tumor Protein (TCTP) Gene From Litopenaeus Vannamei And Its Over-expression In Cultured Primary Shrimp Cells

Although many efforts have been done in the shrimp cell culture, up to date, noimmortalized shrimp cell line has been successfully established and only primaryshrimp cell culture can be obtained and maintained successfully. Continuous shrimpcell line can provide us a useful tool for the study of the biology and pathogenicmechanism of shrimp virus as well as for the preparation of shrimp virus vaccine. Ithas been reported that over-expression of exogenous oncogenes in mammalian cellscan successfully induce the immortalization transformation of the cells and establish acontinuous cell line. Translationally controlled tumor protein (TCTP) is reported asgrowth-related protein and plays a key role in several cellular processes including cellgrowth, apoptosis and cell reprogramming. Therefore, the main purposes of this studyare to clone the TCTP gene from Litopenaeus vannamei and construct the eukaryoticexpression vector, and to analyze the role of over-expressed TCTP on theimmortalization transformation of shrimp primary cell culture by lipofection method.The obtained data will lay a technical foundation for the immortalizationtransformation of shrimp primary cells.In the present study, we firstly successfully cloned the full-length cDNA of theopen reading frame (ORF) of TCTP gene from Litopenaeus vannamei by RT-PCR,named as Lv-TCTP. The obtained Lv-TCTP fragment is507bp in size, encoding168amino acids. Second, using the plasmid pCX-eGFP as a template, the ORF ofenhanced green fluorescent protein (eGFP) was amplified by PCR. Then Lv-TCTPgene was directional inserted into the eukaryotic expression vectorpcDNA3.1-V5/HisA flanking with BamHI and EcoRI to construct pcDNA3.1-TCTPrecombinant plasmid, followed by the directional insertion of eGFP gene into the recombinant plasmid pcDNA3.1-TCTP flanking with KpnI and BamHI to constructthe eGFP fusion protein expression vector of pcDNA3.1-eGFP/TCTP.The fusion protein expression vector plasmid pcDNA3.1-eGFP/TCTP was thentransformed into the mammalian cells (Plat-GP) by lipofection method. Twenty fourhours later, obvious green fluorescence was observed in the transformed Plat-GP cellsunder the inverted fluorescence microscope, indicating that the constructed expressionplasmid of pcDNA3.1-eGFP/TCTP can be expressed effectively in plat-GP cells.When the same expression plasmid was transformed into the primary culture cellsderived from the lymphoid-like tissues (Oka organ) of shrimp Litopenaeus vannameiby the same lipofection method, however, no green fluorescence was observed in thetransformed shrimp cells, showing that the above-mentioned expression plasmid wasnot be expressed effectively in shrimp cells. The genomic DNA and total RNA werethen extracted from the transformed shrimp primary cells at96hours aftertransfection, respectively, and PCR amplification was performed to investigate theintroduction and transcription of foreign eGFP gene in the shrimp cells usingeGFP-specific primers. The obtained results showed that eGFP-specific fragmentcould be amplified using genome DNA as a template, but not using the cDNAderived from total RNA as a template, indicating that the expression plasmidpcDNA3.1-eGFP/TCTP had been successfully introduced into the shrimp cells, butnot been transcribed successfully. This is in agreement with the results of greenfluorescence observation in the transfected shrimp cells.The promoter of the expression plasmid pcDNA3.1-eGFP/TCTP iscytomegalovirus promoter (CMV), the results from the transformation experimentsusing this expression plasmid showed that the activity of CMV promoter is strong inmammalian cells, but weak in shrimp cells. Therefore, we cloned the promoter regionof TCTP gene from Litopenaeus vannamei (Ptctp) which was605bp in size, located inthe3’UTR of Lv-TCTP gene (at the position from-3to-617). Then the Ptctpwasdirectionally inserted into the expression vector of pcDNA3.1-eGFP/TCTP at theposition between CMV and eGFP flanking with BamHI and EcoRI and the expressionvector plasmid pcDNA-Ptctp-eGFP/TCTP was successfully construct. Then the expression vector plasmid of pcDNA-Ptctp-eGFP/TCTP was transformed into thecultured shrimp primary cells by lipofection method to analyze the transcriptionalactivity of Ptctpin shrimp cells. After24hours, green fluorescence signal could bedetected under the fluorescence microscope. The obtained results showed that, thePtctp promoter from Litopenaeus vannamei can effectively activate the transcriptionand expression of exogenous genes in shrimp cells. And also, the genomic DNA andtotal RNA were then extracted from the transformed shrimp primary cells at96hoursafter transfection, respectively, and PCR amplification was performed to investigatethe introduction and transcription of foreign eGFP gene in the shrimp cells usingeGFP-specific primers. The obtained results showed that eGFP-specific fragmentscould be amplified from the above-mentioned two templates, indicating that theexpression plasmid pcDNA3.1-eGFP/TCTP had been successfully introduced into andtranscribed in the shrimp cells. This is also in agreement with the results of greenfluorescence observation in the transformed shrimp cells.In order to examine the effect of fused eGFP protein on the biological function ofTCTP, a non-fusion protein expression vector pcDNA3.1-Ptctp-TCTP, whichexpressed TCTP alone, containing shrimp promoter Ptctpbut not eGFP gene, wasconstructed. Plasmids of pcDNA-Ptctp-eGFP/TCTP and pcDNA3.1-Ptctp-TCTP weretransformed into the primary cultured shrimp lymphoid cells respectively and theeffects of over-expressed eGFP/TCTP and TCTP on the immortalizationtransformation of shrimp cells were compared. The obtained results indicated that,compared with the control group, the shrimp cells transformed with theabove-mentioned two expression plasmids exhibited better cellular status with highercell survival rate and relative stubby shape, indicating an effect on cell transformation;but no obvious difference in cellular morphology and survival rate was observed inthe shrimp cells transformed with pcDNA-Ptctp-eGFP/TCTP andpcDNA3.1-Ptctp-TCTP, inferring that the fused eGFP did not affect the fold andconformation of TCTP. And also, the transformed shrimp cells could be maintained over40days in a healthy status. However, we failed to get the immortalized shrimpcells due to microorganism contamination.Taken together, all the obtained data in the present study have laid a technicalfoundation for the future work on the immortalization transformation of shrimpprimary cells and biological functions of shrimp genes.