日本血吸虫Sj26gst mRNA候选疫苗的免疫原性研究

Immunogenicity of Schistosoma japonicum Sj26gst mRNA vaccine candidate

目的制备日本血吸虫谷胱甘肽S⁃转移酶相对分子质量(Mr)26000亚基(Sj26gst)mRNA并评价其免疫原性。方法根据Sj26gst编码区序列(NC_002544.1),用人类常用的密码子优化Sj26gst编码序列,两端加β球蛋白3′和5′非编码区(UTR),合成后连接至pcDNA3.1(+),构建pcDNA3.1/Sj26gst质粒,以线性化的pcDNA3.1/Sj26gst质粒为模版,体外转录Sj26gst mRNA。用脂质体将Sj26gst mRNA转染至HeLa细胞中,转染后2、6、12、24、48 h取HeLa细胞,免疫荧光定量PCR(qRT⁃PCR)检测Sj26gst mRNA的相对水平,蛋白质免疫印迹(Western blotting)检测Sj26GST蛋白的表达水平。雌性BALB/c小鼠随机均分为Sj26gst mRNA组、pcDNA3.1/Sj26gst组、pcDNA3.1组和PBS组(每组6只),分别于小鼠左后腿股四头肌肌内注射100μg的Sj26gst mRNA、pcDNA3.1/Sj26gst质粒、pcDNA3.1质粒和PBS(100μl),共免疫3次,每次间隔2周。分别采集免疫前(0),首次免疫后2、4、6、8周小鼠尾静脉血,制备血清,ELISA检测血清Sj26GST特异性IgG抗体水平。首次免疫后8周,无菌分离小鼠脾组织,制备脾淋巴细胞悬液,37℃培养48 h后,加入10μl细胞增殖试剂培养4 h后测定脾细胞刺激指数,ELISA法检测脾淋巴细胞上清中Th1型[肿瘤坏死因子α(TNF⁃α)、干扰素γ(INF⁃γ)],Th2型[白细胞介素4(IL⁃4)、IL⁃10]和Th17型(IL⁃17)细胞因子的水平。组间比较采用单因素方差分析。结果优化后的Sj26gst编码序列长858 bp,GC含量为63.2%,二级结构的自由能变为-485.6 kcal/mol。电泳结果显示,体外转录的Sj26gst mRNA条带呈单一且无弥散的条带。RT⁃PCR结果显示,Sj26gst mRNA转染后2 h,HeLa细胞Sj26gst mRNA相对水平为6.21±0.83,6 h达到峰值(14.26±1.23),随后下降;Western blotting结果显示,Sj26gst mRNA转染后6、12、24、48 h均检出Mr 26000的Sj26GST蛋白条带。ELISA检测结果显示,首次免疫后8周,Sj26gst mRNA组、pcDNA3.1/Sj26gst组、pcDNA3.1组和PBS组的A450分别为0.85±0.16、0.42±0.21、0.14±0.03和0.11±0.02,Sj26gst mRNA组与pcDNA3.1/Sj26gst组、pcDNA3.1/Sj26gst组与pcDNA3.1组之间差异均有统计学意义(t=16.46、12.35,均P<0.05)。首次免疫后8周,Sj26gst mRNA组、pcDNA3.1/Sj26gst组小鼠脾淋巴细胞增殖指数分别为11.25±1.22、6.37±2.45,高于pcDNA3.1组(2.56±0.38)和PBS组(1.27±0.45)(t=9.98、5.25,均P<0.05)。ELISA检测结果显示,Sj26gst mRNA组小鼠脾淋巴细胞上清中TNF⁃α、INF⁃γ、IL⁃10和IL⁃17的表达水平分别为(756.23±134.35)、(598.46±50.47)、(713.42±118.25)、(301.45±48.23)pg/ml,pcDNA3.1/Sj26gst组分别为(587.26±32.48)、(401.45±46.25)、(386.27±23.75)、(175.24±41.23),均高于pcDNA3.1组的(19.34±2.01)、(35.14±5.25)、(32.11±15.20)、(19.75±7.21)pg/ml和PBS组的(18.75±1.98)、(34.12±4.78)、(29.36±8.72)、(15.34±2.51)pg/ml(t=54.59、19.57、52.36、26.47,均P<0.05)。结论构建了稳定表达Sj26gst mRNA的质粒,Sj26gst mRNA可诱导产生高水平的特异性Sj26GST蛋白抗体并诱导Th1型细胞免疫应答。

Objective To evaluate glutathione S⁃transferase subunit of relative molecular mass(Mr)26000(Sj26gst)mRNA of Schistosoma japonicum and assess its immunogenicity.Methods The Sj26gst coding sequence was optimized by the commonly used human codons according to the Sj26gst coding region sequence(NC_002544.1).The gene was synthesized by adding the 3′and 5′non⁃coding regions(UTR)of beta⁃globulin at both ends,and then con⁃nected to pcDNA3.1(+)to construct the linear pcDNA3.1/Sj26gst plasmid which was used for transient transcription of Sj26gst mRNA in vitro.The Sj26gst mRNA was transfected into HeLa cells by using liposomes.The HeLa cells were collected to detect the relative expression level of Sj26gst mRNA by quantitative immunofluorescence PCR(qRT⁃PCR)and examine the expression level of Sj26GST protein by Western blotting at 2,6,12,24,48 hours after the transfec⁃tion.Female BALB/c mice were randomly divided into four groups:Sj26gst⁃mRNA group,pcDNA3.1/Sj26gst group,pcDNA3.1 group and PBS group(6 mice in each group).Mice were immunized three times at two⁃week interval with 100μg of Sj26gst mRNA,pcDNA3.1/Sj26gst plasmid,pcDNA3.1 plasmid,or PBS(100μl),respectively.Blood samples were collected from the tail vein before immunization(0 week)and at 2,4,6 and 8 weeks after the first immuniza⁃tion for serum preparation which was used to detect SJ26GST specific IgG antibody levels by ELISA.At 8 weeks post⁃immunization,the mice spleen tissue was collected aseptically to prepare spleen lymphocyte suspension,which was cul⁃tured at 37℃for 48 h,subsequently,10μl CCK8 reagent(cell counting kit⁃8)was added for culturing additional 4 h to determine the spleen cell stimulation index.The levels of cytokines Th1(TNF⁃α,INF⁃γ),Th2(IL⁃10,IL⁃4)and Th17(IL⁃17)in the lymphocyte supernatant were assayed by ELISA.One⁃way analysis of variance was used for inter⁃group comparisons.Results The optimized coding sequence was 858 bp in length,the GC content was 63.2%,and the free energy change of the secondary structure was-485.6 kcal/mol.The in vitro transcribed Sj26gst mRNA band was a single and non⁃dispersive band.The RT⁃PCR results showed that the relative expression level of Sj26gst mRNA in HeLa cells is(6.21±0.83)at 2 h after Sj26gst mRNA transfection,and its peak level was(14.26±1.23)at 6 h,followed by a decrease.The western blotting results showed that Sj26GST protein bands of Mr 26000 were detected at 6,12,24 and 48 hours after Sj26gst mRNA transfection.The ELISA results showed that the A450 value of Sj26gst mRNA group,pcDNA3.1/Sj26gst group,pcDNA3.1 group and PBS group were 0.85±0.16,0.42±0.21,0.14±0.03 and 0.11±0.02,at eight weeks after the first immunization,respectively.There were statistically significant differences between Sj26gst mRNA group and pcDNA3.1/Sj26gst group,pcDNA3.1/Sj26gst group and pcDNA3.1/Sj26gst group(t=16.46,12.35;all P<0.05).The ELISA results showed that the TNF⁃α,INF⁃γ,IL⁃10,and IL⁃17 expression levels in the supernatants of splenocytes from Sj26gst mRNA⁃immunized mice were(756.23±134.35),(598.46±50.47),(713.42±118.25),and(301.45±48.23)pg/ml,respectively.In the PcDNA3.1/Sj26gst group,the corresponding levels were(587.26±32.48),(401.45±46.25),(386.27±23.75)and(175.24±41.23)pg/ml.These levels were higher than those in the pcDNA3.1 group[(19.34±2.01),(35.14±5.25),(32.11±15.20),(19.75±7.21)pg/ml]and the PBS group[(18.75±1.98),(34.12±4.78),(29.36±8.72),(15.34±2.51)pg/ml](t=54.59,19.57,52.36,26.47;all P<0.05).Conclusion The Sj26gst mRNA plasmid with stable expression of Sj26GST was constructed.The Sj26gst mRNA could induce high level of specific antibodies against Sj26GST and stimulate Th1 cellular immune response.