密码子优化增强新城疫病毒DNA疫苗的表达水平和免疫效果

为提高新城疫病毒（Newcasti Disease Virus，NDV）F基因DNA疫苗的表达量，增强其免疫效果。按照鸡体内偏嗜性密码子人工合成了NDV F48E9株的F基因optiF，插入到真核表达载体pVAX1中获得pVAX1-optiF。将F48E9株的F基因pVAX1-F与pVAX1-optiF分别转染COS-7细胞，72h后间接免疫荧光和Western blot检测细胞中瞬时表达的F蛋白。将质粒pVAX1、pVAX1-F和pVAX1-optiF以200μg／只的剂量分别股四头肌多点注射18只2周龄SPF鸡，2周后加强免疫1次，同时设立PBS对照。二免2周后每组取12只鸡以100EID50的F48E9株NDV强毒进行攻毒，评价这2种DNA疫苗的免疫效果。结果表明，F基因密码子的优化可显著提高NDVDNA疫苗诱导的保护性体液免疫和细胞免疫应答水平，攻毒后所有pVAX1-optiF免疫鸡均获得保护（12／12），而pVAX1-F组保护率只有17％（2／12）。DNA疫苗的免疫效果与抗原基因的表达量及表达抗原的免疫原性有直接关系。与未经修饰的F基因相比，修饰后的F基因体外瞬时表达水平明显提高。

OPTIMIZATION OF CODON USAGE OF F GENE ENHANCED EFFICACY OF NEWCASTLE DISEASE VIRUS DNA VACCINE

In order to enhance the immunogenicity of DNA vaccines, the F gene of the vaccine strain NDV F48F4 was optimized according to the chicken bias codon usages. The optimized F gene （optiF） and the wild-type F gene were inserted into the pVAXlvector, respectively, and then two recombinant plasmids pVAXl-optiF and pVAX1-F were generated. The F protein transiently expressed in COS-7 cells was detected in indirect immunofluorescence and Western blot at 72 h post transfection. Results showed that the in vitro expression of optiF was considerably increased in comparison with that of the respective wide-type sequence in the same vector. Each of two-week-old SPF chickens was inoculated by multi-point injection with 200 t~g of plasmids pVAX1, pVAXl-F and pVAXl-opti F, respectively. The control group was injected with PBS alone. A booster immunization was administered with the same dose two weeks later. All chickens were challenged additional two weeks later with 10^5 EID50 of the NDV F48 E9 velogen strain and their morbidity and mortality were calculated. Blood samples were collected weekly for detecting the antibody against the F protein. Peripheral blood lymphocytes were isolated and examined for the composition of CD4 ＋ ,CD8 ＋ and TCRγδ T cells. Splenic lymphocytes proliferation assay was performed to measure cell-mediated immunity. Chickens vaccinated with pVAXl-optiF more quickly developed antibody response to F protein, produced significantly enhanced stimulation index and marked increase in percentage of CD4 ＋ , CD8 ＋ and TCRγδ T cells as compared to those chickens vaccinated with pVAX1-F. In addition, chickens in the optiF vaccine group were completely protected from the virus challenge. These results indicated that codon optimization of F gene significantly enhanced the efficacy of the NDV F48 E9 strain DNA vaccine.