猪伪狂犬病病毒双基因缺失gI^-／gE^-／PRV SA738灭活疫苗的安全性与免疫效力

在构建gE和gI双基因缺失猪伪狂犬病毒（Pseudorabiesvirus，PRV）SA738株的基础上，以绿色荧光蛋白为标志，将gI^-／gE^-／PRVSA738转柒BHK-21细胞，通过蚀斑法得到纯化的gI^-／gE^-／PRVSA738。毒价检测结果显示，TCID50由7．25下降至5．75，表明该病毒已被弱化，与预期结果相当。将该病毒灭活后，以不同滴度接种无母源抗体仔猪及妊娠母猪，安全性检测表明，gI^-／gE^-／PRVSA738对仔猪体温、生长，对妊娠母猪产仔及仔猪健康状况均无影响。随后将灭活的gI^-／gE^-／PRVSA738与弗氏佐剂混合乳化，分别接种家兔与仔猪，免疫后7、14、21、28d采血分离血清。经ELISA检测，家兔免疫后14d、仔猪免疫后7d均显示阳性，对照组均为阴性。抗体中和试验表明，家免免疫后14d抗体水平比7d显著升高，但至21d，抗体升高不明显；仔猪抗体水平至28d，中和抗体水平达到1：262．23，与21d相比差异极显著。免疫后28d，采用100LD50 PRV强毒攻击动物，进行免疫保护性试验，结果显示，家兔免疫组只有一只出现轻微的一过性临床症状，未出现死亡，而对照组出现了奇痒等较重的临床症状，且于5d后全部死亡；仔猪对照组攻毒后2d，出现了精神高度沉郁、不食、呕吐与腹泻等症状，并于5d后死亡；免疫组攻毒后3d，出现轻微食欲不振、精神萎靡、腹泻等症状，且于攻毒后6d恢复正常，免疫保护率为100％。以上结果表明，该gI^-／gE^-／PRVSA738-突变株有效诱导了机体的保护性免疫应答。

The Safety and efficacy of gI^-/gE^- negative pseudorabies virusSA738 strain vaccine

The gI^-/gE^- negative PRV SA738 strain was transfected to BHK-21 cells and depurated by plaque with the green fluorescent protein as a symbol. The TCID50 decreased from 7.25 to 5.75,indicating that the virulence of PRV SA738 strain was attenuated,because the knockout of gI/gE gene. The pregnant sows and piglet without maternal antibody were inoculated with inactivated gI^-/gE^- /PRV SA738. The results indicated that the gI^-/gE^-/ PRV SA738 did not impact the body temperature and growth of piglet,and was safe for littering of pregnant sows and health of piglet. The rabbits and piglets were inoculated with inactivated gI^-/gE^-/PRV SA738 mixed and Freund adjuvant respectively. The serum was prepared and analyzed on 7,14,21 and 28 d after inoculation. The results showed that the serum displayed masculine on 14 d and 7 d respectively after the rabbits and the piglets were inoculated,but the controls were negative. Subsequently, the level of antibody was determined by antibody neutralization test. On 14 d,the antibody level of rabbits was obviously higher than that of 7 d. But by the end of 21 d,it was not remarkably high. The antibody of level of piglets was 262.23 on 28 d,obviously higher than 21 d. The rabbits and piglets were attacked with 100LD50 virulent PRV after 21 d and 28 d respectively. The results showed that only one displayed feeble clinical symptom,but the symptom of the control was very serous and died on 5 d. The piglets displayed loss of appetite,listlessness and diarrhea, but recovery on 6d,indicating the rate of protection of gI^-/gE^-/ PRV SA738 is 100 %. However, the piglet control exhibited depressed, vomiting, diarrhea, and died on 5 d after attacked. Collectively, the gI^-/gE^-/PRV SA738 strain induced the efficient protective immune response with stronger and longer antibody level.