Expression Of Porcine Circovirus Type 2 ORF2 Antigen Gene In Chlamvdomonas Reinhardtii And Tobacco Chloroplasts

In recent years,plant expression systems have become more and more prominent in the field of genetic engineering research,plant chloroplast genetic transformation system has become a research hotspot of plant bioreactor due to its unique advantages.Using plant chloroplast as expression system can realize site-specific integration of exogenous genes and co-expression of multiple genes;plant chloroplasts contain multiple genomic copies,the expression efficiency of exogenous genes will be significantly improved;In addition,chloroplasts belong to maternal inheritance,exogenous genes will not escape with pollen transmission,and the biological environment is safe.Chlamydomonas reinhardtii has short growth cycle and is easy to be cultured;it contains a large cup-shaped chloroplast which accounts for about 70%of the cell volume,genetic manipulation is simple and feasible,so it is an ideal place for chloroplast transformation.Nicotiana tabacum has large biomass,tissue culture and regeneration system is mature,it has been widely used in the study of chloroplast genetic transformation of higher plants.Therefore,the major antigen gene ORF2 of PCV2 was recombined and introduced into chloroplasts of C.reinhardtii and tobacco for expression,in order to provide theoretical and technical support for exploring new production routes of important medicinal proteins.The experiment is divided into the following two parts:(1)Recombination and expression of PCV2 major antigen gene ORF2 in C.reinhardtii chloroplastIn this experiment,the ORF2 antigen gene C.reinhardtii chloroplast expression vector pCR02 containing the optimized synthesis ORF2 and aadA gene was constructed.The expression vector pCR02 was introduced into C.reinhardtii chloroplast by biolistic bombardment method.After resistance screening,13 resistant transformants were obtained.PCR results showed that the target bands of ORF2 exogenous gene appeared in the PCR products of 4 resistant transformants,and ORF2 antigen gene was correctly integrated into the specific site of C.reinhardtii chloroplast genome.In addition,RT-PCR,SDS-PAGE,Western-blot and ELISA tests proved that Cap protein encoded by ORF2 antigen gene was expressed and had immunogenicity.(2)Recombination and expression of PCV2 major antigen gene ORF2 in tobacco chloroplastIn this experiment,ORF2 antigen gene of PCV2 was optimized and synthesized according to the preference of codon expression in tobacco chloroplast genome.The fusion promoter PrrnPclpPrbcL and terminator TpsbA from tobacco chloroplast genome were selected as the regulation sequences of the ORF2 gene,tobacco chloroplast genome sequence 16S-trnI-trnA-23S was selected for homologous recombination and aadA resistance gene was used for marker selection.Finally,the ORF2 antigen gene tobacco chloroplast expression vector pNK-a03 was constructed.The expression vector pNK-a03 was introduced into tobacco chloroplast by gene gun.After screening for spectinomycin resistance,20 resistant tobacco plants were obtained.PCR results showed that the target products of ORF2 gene were detected in 3 resistant plants.Further,the primers which complementary to the tobacco chloroplast homologous recombination sequence and ORF2 gene sequence respectively were used to detect the insertion site of ORF2 exogenous gene,the results showed that the target gene has been correctly integrated into the specific site of tobacco chloroplast genome;RT-PCR,SDS-PAGE,Western-blot and ELISA tests also proved that ORF2 antigen gene was normally transcribed in tobacco chloroplast and Cap protein was effectively expressed.