以小鼠模式研發第二型豬環狀病毒DNA疫苗

Abstract

本研究的目的是以小鼠模式評估PCV2 DNA疫苗的免疫原性。有學者研究指出以原始的PCV2 ORF2轉譯出來的核衣蛋白(Cap)其轉形量低，且不論是在原核或真核系統都不易偵測到PCV2核衣蛋白的表現量。文獻指出刪除核衣蛋白的核定位訊號(nuclear localization signal；NLS)區域，能使修飾過的核衣蛋白具有較高的表現量。因此，本研究將依此建構PCV2核衣構築，分別建構含有人類細胞巨大病毒啟動子之真核表現載體(pcDNA3.1/V5-His-TOPO)，以及含有T7 RNA聚合酶啟動子之大腸桿菌表現載體(pET 21)，而構築出重組表現質體，並藉由西方墨點法來確認細胞內蛋白的表現且進一步評估在小鼠模式的的免疫原性。共設計四組不同的DNA構築，分別為(1)PCV2a亞型原始cap gene(pcDNA-2a)；(2)PCV2b亞型原始cap gene(pcDNA-2b)；(3)PCV2a 亞型修飾過的cap gene(pcDNA-Δ2a)；(4)PCV2b 亞型修飾過的 cap gene(pcDNA-Δ2b)。將上述四組構築載入真核表現系統之載體(pcDNA3.1/V5-His TOPO)內，作為DNA疫苗使用，再將此四組疫苗以100 μg/次肌肉注射的方式免疫小鼠四次，每次間隔兩週，藉以評估其誘發之免疫反應，於實驗期間收集小鼠之血清作為PCV2 特異性抗體檢測之用，並於小鼠犧牲時收集其脾臟細胞作為淋巴細胞增殖反應檢測之用。結果顯示以大腸桿菌表現蛋白誘導之蛋白質電泳，經 Coomassie blue 染色可見以0.1和 0.5 mM IPTG誘導之pET-Δ2a和 pET-Δ2b 有明顯之蛋白質表現，目標產物分別為24 kDa 及22 kDa。而在西方墨點法僅有 pET-Δ2a有微量之蛋白質表現；而以間接螢光免疫分析上述四組構築真核表現載體轉染至PK-15 細胞質內皆有陽性的綠色顆粒樣螢光訊號，經Coomassie blue染色和西方墨點法分析可見四組載體皆呈現微量之蛋白質表現。而經DNA接種四次之小鼠，MTS assay呈現微弱之淋巴球增殖反應，且各組間並無顯著差異(p>0.05)，而以流式細胞儀分析各組間雖無顯著差異(p>0.05)，但各組內之陽性對照組(Concanavalin A)與病毒刺激組和未處理組有顯著差異(p<0.05)。以商用ELISA套組檢測各組小鼠血清中IgG抗體均為陰性，將注射構築之四組DNA疫苗以間接螢光免疫分析(IIFA)，在螢光顯微鏡下觀察到各組小鼠血清IgG抗體亦皆為陰性。顯示雖然在各構築有可見之蛋白表現，唯在活體測試的小鼠模式，並未激發特異之免疫反應。

The capsid (Cap) protein encoded by the PCV2 ORF2 gene may be an potential candidate for vaccine development. The objective of the study was to evaluate the immunogenicity of porcine circovirus type 2 (PCV2) DNA vaccines in mice model. Previously studies showed native PCV2 ORF2 encoded capsid protein(Cap)demonstrated low transformation yield and almost undetectable PCV2-Cap expression levels in either prokaryotic or eukaryotic system. It has been noted that deletion of the nuclear localization signal (NLS) region of the Cap protein led to a higher level expression of the dCap protein. Therefore, these constructs were performed in the present study. We have constructed the pcDNA3.1/V5-His-TOPO, a eukaryotic expression vector containing the human cytomegalovirus promoter,and a pET 21,a Escherichia coli expression vector containing the T7 RNA polymerase promoter, respectively. Further,the recombinant expression plasmids were constructed. Protein expression and its immunogenicity was confirmed by Western blot analysis and mice model. Four DNA constructs included (1) PCV2a native cap gene(pcDNA-2a); (2)PCV2b native cap gene (pcDNA-2b);(3)PCV2a modified cap gene (pcDNA-Δ2a);(4)PCV2b modified cap gene (pcDNA-Δ2b).One hundred μg of constructed DNA were injected into mice intramuscularly for four times at a 2-week interval. Serum samples were collected at various designated time points for the measurement of PCV2-specific antibodies and splenocytes were isolated at time of sacrifice for lymphocyte blastogenesis assay. The results showed that Coomassie blue staining of cell pellets of pET-Δ2a and pET-Δ2b were noted in E.coli system after 0.1 and 0.5 mM IPTG induction. The size of each expression protein were 24 kDa and 22 kDa respectively, and only pET-Δ2a showed weak expression in the Western blot analysis. However, weak results were both noted in the Coomassie blue staining and Western blot analysis in all recombinant subunit protein expressed in transfected PK-15 cells. In addition, weak intracytoplasmic positive signals were noted in PK-15 cells transfected with various recombinant constructs by IIFA. The immunogenicity assay of four constructs in mice model reveals no significant cell-mediated response in MTS assay and flow cytometry, and no significant humoral response in blocking ELISA assay and IIFA.