Research on immune effect of two gene vaccines containing brZPC′and brLDHC_4′when in combined inoculation

ZPC and LDHC_4 play a key role in the process of recognition between sperm and egg or sperm movement,respectively.In this study,partial cDNA sequences of ZPC and LDH-C_4 of Microtus branditi(brZPC and brLDHC_4,respectively) were cloned by RT-PCR,and directly inserted into pCR3.1 vector to construct two gene vaccines(pCR3.1-brZPC' and pCR3.1-brLDHC'_4).pCR3.1-brZPC' and pCR3.1-brLDHC,' could express corresponding proteins in transiently transfected CHO cells.The adult female BALB/c mice were inoculated with the recombinant vaccine alone on in combination.The immunized mouse can produce specific antibody that recognizes the corresponding recombinant protein expressed by BL21 in vitro.Moreover,antibodies produced by combinedly immunized mouse were specific and direct,with no inhibitory effect between two vaccines observed when in combined inoculation.The test of cytokines indicated that the expression of IFNγin pCR3.1-brZPC'- and combinedly inoculated mouse increased obviously and the expression of IL2 in pCR3.l-brLDHC'_4- and combinedly inoculated mouse increased obviously,while the expression of IL4 in pCR3.1-brZPC,pCR3.l-brLDHC'_4 and combinedly inoculated group increased obviously.It was suggested that the combined inoculation with pCR3.1-brZPC' and pCR3.l-brLDHC'_4 could induce both humoral immune response and CTL response.In some sense,the combined inoculation may achieve a better contraceptive effect.The results also showed that,when used alone or in combination,these two recombinant vaccines did not do much harm to the follicular development of immunized mouse.So the combined inoculation with these two recombinant vaccines could be a better way to immunocontraception.This study may provide a theoretical basis for the following tests on antifertility in vivo.

ZPC and LDHC4 play a key role in the process of recognition between sperm and egg or sperm movement, respectively. In this study, partial cDNA sequences of ZPC and LDH-C4 of Microtus branditi （brZPC＇ and brLDHC4＇, respectively） were cloned by RT-PCR, and directly inserted into pCR3.1 vector to construct two gene vaccines （pCR3. 1-brZPC＇ and pCR3. 1-brLDHC4＇）. pCR3. 1-brZPC＇ and pCR3. 1-brLDHC4＇ could express corresponding proteins in transiently transfected CHO cells. The adult female BALB/c mice were inoculated with the recombinant vaccine alone on in combination. The immunized mouse can produce specific antibody that recognizes the corresponding recombinant protein expressed by BL21 in vitro. Moreover, antibodies produced by combinedly immunized mouse were specific and direct, with no inhibitory effect between two vaccines observed when in combined inoculation. The test of cytokines indicated that the expression of IFN7 in pCR3. 1-brZPCC and combinedly inoculated mouse increased obviously and the expression of IL2 in pCR3.1-brLDHC4＇- and combinedly inoculated mouse increased obviously, while the expression of IL4 in pCR3. 1-brZPC＇, pCR3. 1-brLDHC4＇ and combinedly inoculated group increased obviously. It was suggested that the combined inoculation with pCR3.1-brZPC＇ and pCR3.1-brLDHC4＇ could induce both humoral immune response and CTL response. In some sense, the combined inoculation may achieve a better contraceptive effect. The results also showed that, when used alone or in combination, these two recombinant vaccines did not do much harm to the follicular development of immunized mouse. So the combined inoculation with these two recombinant vaccines could be a better way to immunocontraception. This study may provide a theoretical basis for the following tests on antifertility in vivo.