Transient And Rapid Expression Of The Major Surface Antigenic Gene1Peptides Of Toxoplasma Gondii In Nicotiana Plants

Transgenic plant vaccine is convenient to use and with low cost and high safety. At present, many plant vaccines have been expressed in transgenic plants successfully, and some transgenic plant vaccines against viral and bacterial diseases have entered clinical trials. It shows its broad application prospects.Toxoplasma gondii is an opportunistic protozoan parasite which can infect both humans and animals. It can cause zoonosis toxoplasmosis. It is a serious threat to pregnant women, children, individuals with immunodeficiency and the development of animal husbandry. Therefore, exploration of a cheap transgenic plant vaccine against Toxoplasma gondii is a predomiant new way to block the transmission from the infected animals to human beings and easily utilized for control of animal toxoplasmosis.Based on the rapid high-yield plant transient protein expression, the SAG1peptides gene of Toxoplasma gondii was introduced into Nicotiana plants by infiltrating with Agrobacterium tumefaciens LBA4404. Through identification of Nicotiana leaf total soluble protein, we expect to obtain rapid high-yield expression of SAG1peptides of Toxoplasma gondii with good immune responsiveness. This study forms a basis for studying Toxoplasma gondii plant vaccine. Chapter I A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditionsObjectiveThis study aims to develop a hydroponic Nicotiana cultivation system for rapid high-yield transient expression of recombinant proteins under laboratory conditions and forms a basis for implementing small scale transgenic plant vaccines research easily in the general molecular biology laboratory.Methods1. Based on the hydroponic cultivation system, the impact of the tightness of the matric, temperature regime, relative humidity and day-length on Nicotiana growth were investigated. When Nicotiana grew strong, uniform and had developed root, green leaf, the growth conditions were chosen as the optimal candidate.2. We chose to use the green fluorescent protein (GFP) and the Geminiviral plant transient expression vector as the model protein/expression vector for this investigation. Specifically, we examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by UV, Western blot and ELISA analyses.3. ELISA data were processed by SPSS13.0. Two Nicotiana species groups were overall compared using a factorial design analysis of variance. The density and time of Agrobacterium infiltration groups, and the post-infiltration growth period groups were compared within the groups using One-way ANOVA analysis. Two Nicotiana species groups in each density, time of Agrobacterium infiltration and post-infiltration growth period point were compared using Independent-Samples T Test analysis. P<0.05indicates that the difference has statistics significance.4. Fresh Nicotiana leaves from the hydroponic system were weight, and average leaves weight was utilized for biomass comparison evaluation. Biomass of two Nicotiana varieties were compared by the T-Test analysis, P<0.05indicates the difference has statistics significance. Results1. We have established a hydroponic cultivation system that allows the robust growth of Nicotiana in the artificial climate box. The tightness of the matric holding tobacco root was moderate, which was required to achieve the state of holding group and touching scattered. Improved Hoagland nutrient fluid was used for providing nutrition. In addition, our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of9000LX/layer, a light cycle of16hr day/8hr night, a temperature regime of28℃day/21℃night, the air pump for synchronous airing, ventilation of10min/h, and a relative humidity of80%would support the optimal Nicotiana growth that they grew strong, uniform and had developed root, green leaf.2. After agroinfiltration with pBYGFPDsRed.R/LBA4404, the optimal GFP expression was observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants. Our study showed that an optimal GFP expression could be achieved in both Nicotiana species leaves4days after they were infiltrated by Agrobacterium with an OD600of0.8. We also found that leaves from6-week-old N. benthamiana plants and5-week-old N. tobaccum (cv. Yuyan No.5) plants were the optimal material for Agroinfiltration. Our results indicated that at a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N.benthamiana.ConclusionBased on the Geminiviral expression vector pBYGFPDsRed.R, we have established a hydroponic cultivation system that allows the robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box. It has been confirmed that N. tobaccum (cv. Yuyan No.5) would be used as a good plant for rapid high-yield transient expression of recombinant proteins.Chapter II Gene cloning and transient and rapid expression of SAG1peptides of Toxoplasma gondii in Nicotiana plantsObjectiveTo rapid express SAG1peptides of Toxoplasma gondii in Nicotiana plants. Methods1. SAG1of Toxoplasma gondii was chosen as the candidate. Nicotiana codon usage frequency table was obtained from www.kazusa.or.jp/codon (codon use frequency network) for further codon optimization. Based on the literature, some dominant peptides of SAG1containing the T and B cell epitopes were chosen to construct the target candidate and analyzed by BioSun software. Then the target SAG1peptides fragments were optimizated by using Nicotiana favorite codons. The optimized SAG1peptides genes were named as P30-1and P30-II. P30-1was inserted in the middle of HBc and named as HBc-P30-1. P30-II was inserted at the C end of HBc and named as HBc-P30-II. After analyzed by GenScript Rare Codon Analysis Tool, they were optimizated. His-Tag protein gene sequence added to the end of HBc-P30-1and P30-II. HBc-P30-1was synthesized into wheat endosperm cell free expression vector pIVEX1.4WG and P30-II was synthesized into GenScript standard vector PUC57-simple by GenScript company.2. pIVEX1.4WG-HBc-P30-1was identified by restriction enzymes digestion with EcoRI and HindⅢ, and PUC57-simple-P30-Ⅱ were identified by restriction enzymes digestion with NdeI and SalI. The vectors were also confirmed by sequencing.3. PIVEX1.4WG-HBc-P30-I was digested with BglII, and the long segment was ligated for the intermediate vetor pIVEX1.4WG-HBc. After identification by restriction enzymes digestion, P30-II from PUC57-simple-P30-IIwas subcloned into pIVEX1.4WG-HBc to construct the intermediate vetor pIVEX1.4WG-HBc-P30-II with Sal I and Sma I. It was identified by restriction enzymes digestion and sequencing.4. HBc from pIVEX1.4WG-HBc was subcloned into pBYGFPDsRed.R (containing the gene encoding GFP and DsRed-1) to construct rapid high-yield transient protein expression vector pBYR-GFP-HBc (containing the gene encoding GFP) with Xba I and Sac I. It was identified by restriction enzymes digestion and sequencing. 5. P30-I from pIVEX1.4WG-HBc-P30-1was subcloned into pBYGFPDsRed.R (containing the gene encoding GFP and DsRed-1) to construct rapid high-yield transient protein expression vector pBYR-GFP-HBc-P30-1(containing the gene encoding GFP) with Xba I and Sac I. It was identified by restriction enzymes digestion and sequencing.6. HBc-P30-II from pIVEX1.4WG-HBc-P30-II was subcloned into pBYGFPDsRed.R (containing the gene encoding GFP and DsRed-1) to construct rapid high-yield transient protein expression vector pBYR-GFP-HBc-P30-II (containing the gene encoding GFP) with Xba I and Sac I. It was identified by restriction enzymes digestion and sequencing.7. Rapid high-yield transient protein expression vectors containing the gene encoding SAG1peptides of Toxoplasma gondii were delivered to Nicotiana plants by infiltrated with Agrobacterium tumefaciens LBA4404. After agroinfiltration with rapid high-yield transient protein expression vectors/LBA4404, TSP of Nicotiana leaf were extracted on the fourth day. Plant-derived SAG1peptides of Toxoplasma gondii were then analyzed by Western blot using anti-SAG1McAb(Y3A8) and His-Tag (2A8) Mouse mAb.Results1. P30-I and P30-II had several T, B cell epitopes. The optimized SAG1peptides of HBc-P30-I and HBc-P30-II could be predicted to express well in tobacco plants by GenScript Rare Codon Analysis Tool.2. The recombinant intermediate vectors and rapid high-yield transient protein expression vectors were confirmed to have the inserts with expected length of the target fragments by enzyme digestion and sequencing.3. HBc-P30-II was successfully expressed in N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants. The specific reaction band was detected at approximately46.4kDa size by Western blot.ConclusionSAG1peptides genes were designed successfully. The intermedia vectors pIVEX1.4WG-HBc、pIVEX1.4WG-HBc-P30-II and rapid high-yield transient protein expression vectors pBYR-GFP-HBc, pBYR-GFP-HBc-P30-I、 pBYR-GFP-HBc-P30-II were constructed. HBc-P30-II were rapidly expressed in N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants.