Li Hui, Li Rong, Zhong Sen, Ren Hong, Deng Cun-liang, Shi Xiao-ling, Wang Ming-yong
Department of Infectious Disease, Affiliated Hospital of Luzhou Medical College, Luzhou 646000, China.
Zhonghua Jie He He Hu Xi Za Zhi. 2006 Apr;29(4):261-5.
To construct and express a chimeric Mtb8.4 with signal peptide (MS)/hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines.
The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 (pMSI) recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-7 cells were transfected with pMSI constructs by cationic liposome. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture supernatant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte (CTL) induction.
The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-gamma and IL-2 titers were (1,521 +/- 48) ng/L and (755 +/- 41) ng/L in MS/hIL-12 chimeric gene vaccine group, (820 +/- 50) ng/L and (297 +/- 31) ng/L in MS gene vaccine group, (1,487 +/- 40) ng/L and (767 +/- 50) ng/L in BCG group, (121 +/- 16) ng/L and (62 +/- 10) ng/L in vacant vector group, and (48 +/- 16) ng/L and (32 +/- 17) ng/L in PBS group respectively. The levels of IFN-gamma and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group (P < 0.01) and was similar to the BCG group (P > 0.05). The level of IL-4 in BCG group [(91 +/- 11) ng/L] increased significantly as compared to other groups (P < 0.01). When effector-cell-to-target-cell ratio (E:T ratio) were 100:1, 50:1, and 10:1 respectively, the CTL activity was 77.5%, 51.2%, 30.3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups (P < 0.01).
When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.
构建并表达带信号肽的嵌合Mtb8.4（MS）/人白细胞介素12（hIL12）真核表达质粒，研究MS/hIL - 12嵌合基因疫苗的免疫原性。
采用聚合酶链反应（PCR）扩增MS/hIL - 12嵌合基因，并克隆至真核表达载体pCI - neo。通过PCR、限制性内切酶酶切及DNA测序鉴定正确的pCI - neo - MS/hIL12（pMSI）重组质粒。采用阳离子脂质体将pMSI构建体转染COS - 7细胞。48小时后，通过逆转录 - PCR（RT - PCR）检测靶基因的mRNA，采用蛋白质免疫印迹法检测培养上清液及细胞裂解物中的hIL - 12蛋白。C57BL/6N小鼠每隔3周用MS/hIL - 12嵌合基因疫苗接种3次。末次接种后4周，处死3只小鼠以检测细胞因子反应及细胞毒性T淋巴细胞（CTL）诱导情况。
通过多种分子生物学技术证实了质粒构建的准确性。用质粒pMSI转染COS - 7细胞可导致融合蛋白的瞬时表达。MS/hIL - 12嵌合基因疫苗组的干扰素 - γ（IFN - γ）和白细胞介素 - 2（IL - 2）滴度分别为（1521±48）ng/L和（755±41）ng/L，MS基因疫苗组分别为（820±50）ng/L和（297±31）ng/L，卡介苗（BCG）组分别为（1487±40）ng/L和（767±50）ng/L，空载体组分别为（121±16）ng/L和（62±10）ng/L，磷酸盐缓冲液（PBS）组分别为（48±16）ng/L和（32±17）ng/L。MS/hIL - 12嵌合基因疫苗组的IFN - γ和IL - 2水平高于MS基因疫苗组、空载体组和PBS组（P < 0.01），与BCG组相似（P > 0.05）。BCG组的IL - 4水平[（91±11）ng/L]与其他组相比显著升高（P < 0.01）。当效应细胞与靶细胞比例（E:T比）分别为100:1、50:1和10:1时，MS/hIL - 12嵌合基因疫苗组的CTL活性分别为77.5%、51.2%、30.3%，MS基因疫苗组分别为56.2%、37.8%、11.5%，BCG组分别为28.9%、21.4%、9.8%。MS/hIL - 12嵌合基因疫苗组的细胞毒性高于其他组（P < 0.01）。
hIL - 12用于构建嵌合基因疫苗时可提高MS基因疫苗的免疫原性。
