【作者】 贾永清；

【导师】 刘宝全；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2000， 博士

【摘要】 本研究利用RT-PCR方法扩增到一条1.7kb的A/Goose/Guangdong/3/96（H5N1）（GD3）DNA片段，经酶切分析后平端克隆到pUC18中进行了序列测定和分析。结果表明所扩增到的HA基因cDNA全长1731，包含了HA基因完整的开放阅读框架，共编码568个氨基酸，其中信号肽16个氨基酸，HA1 330个氨基酸，HA2 222个氨基酸，并含有起始密码子和终止密码子。GD3与GD1、HK156和HK258核苷酸同源率分别为99.4%、98.6%和98.3%，其受体结合位点和裂解位点的核苷酸与氨基酸组成完全一致。酶切位点的氨基酸序列为一P-Q-R-E-R-R-R-K-K-R-G-L-P-，共有连续5个碱性氨基酸插入。 本试验以LacZ作为报告基因构建了AIV HA基因禽痘病毒转移载体。经转染、蓝斑克隆、筛选和纯化，获得了遗传形状稳定的AIV HA基因重组禽痘病毒。提取感染重组病毒的CEF细胞的DNA进行PCR，扩增出1.7kb的HA基因片段，证明所获得的重组病毒携带有外源目的基因片段。收集纯化的重组病毒CEF细胞培养物无上清液的细胞，用H5亚型AIV多克隆血清作一抗，碱性磷酸酶标记的兔抗鸡IgG为二抗进行Dot-ELISA检测，结果表明重组病毒能在体外的CEF细胞培养中表达HA糖蛋白。Western-blot检测结果证实HA基因表达产物为44kDa（HA1）和23kDa（HA2）的两条带，这两条带都具有与多克隆抗体结合的能力；未发现没有裂解的HAO的存在。 用该重组病毒鸡翅刺种免疫60日龄SPF鸡，免疫3周后，每只鸡用50×LD₅0的GD1株HPAIV攻毒，免疫组无一发病和死亡，攻毒保护率100％。而对照组鸡只全部发病并在攻毒后3～9天相继死亡，发病率和死亡率均为100％。HI抗体效价检测结果表明重组病毒免疫组鸡在免疫后7天即可检测到高效价的HI抗体，抗体效价在免疫后14天达到高峰，随后在较高水平上缓慢下降，攻毒试验对抗体效价及其变化没有影响。灭活苗免疫组鸡在免疫后14天方可检测到HI抗体，抗体效价于免疫后第五周达到高峰。对照组鸡免疫和攻毒前后无HI抗体检出。三组免疫组鸡不同组织器官病毒分离结果全为阴性，而所有对照组鸡都分离到病毒，且肺脏病毒分离率最高（83.9％），脾脏最低（12.9％）。T淋巴细胞亚类检测结果表明：重组病毒免疫后激发了机体的细胞免疫应答，使免疫活性T淋巴细胞活化，攻毒后，免疫鸡CD4⁺、CD8⁺T细胞和TCR1⁺T细胞的数量增高或不变，而对照组鸡CD4⁺、CD8⁺T细胞和TCR1⁺T细胞的数量急剧下降。 总之，通过本试验研究，已经成功地获得了遗传性状稳定，目的基因表达效率高的AIV HA基因重组禽痘病毒，SPF鸡免疫和攻毒试验结果表明，该重组病毒疫苗能全面诱导机体的细胞免疫和体液免疫反应，对强毒攻击产生坚强的免疫保护。本论文基本完成了该基因工程新型疫苗的实验室研究工作，为进一步开展疫苗中试和投入现地使用奠定科学的理论和实验基础，也为我国高致病力禽流感的防制提供了物质储备。更多还原

【Abstract】 Full-length eDNA of haemagglutinin (HA) gene of A/Goose/Guangdong/3/96 (H5N 1) (GD3) was amplified using reverse transcription-polymerase chain reaction (RT-PCR). After identification with neucleotide restriction enzymes,the cDNA was cloned into Sma I site of pUC 18 plasmid and then sequenced. The result of sequencing shows that the eDNA contains whole open reading frame of HA gene, initiation codon and termination eodon. The results of sequence analysis indicated that the cDNA had 11 nucleotide differences and 99.4% of homology in comparison with A/Goose/Guangdong/1/96(H5NI) (GD1), 25 nucleotide differences and 98.6% of homology with AIHongKongIl56I97(H5NI) (HK156), and 30 nucleotide differences and 98.3% of homology with AlChicken/HongKongI2SB/97(1-15N 1) (HK258). Furthermore, The HA of GD3 had the homology of 99.2% with CDI, 98.6% with HK156, and 98.1% with HK258 at amino acid level. They shared the same basic amino acid insert at the cleavage site, and the amino acid sequence is -P-Q-R-E-R-R-R-K-K-R-G-LP-,which indicates that all 4 influenza virus isolates are probably evolved from same ancestor and have similar virulence and biological propertiesIn order to develop a recombinant fowlpox virus (FPV) expressing avian influenza virus (AX) haemagglutinin, the HA cDNA of GD3 and LacZ reporter gene were cloned into pSY538 plasmid and then subcloned into Not I site of pSY68 I plasmid. After construction of transfer vector, it was transfected on CEF cells with FPV F-0l 7. Following 6 cycle screenings, donning and purefieation of blue plaque ,the pure HA-fowlpox virus reeombinants were obtained, and identified by restriction enzyme analysis and PCR. The results showed that the recombinant fowlpox virus contained HA gene of GD3 and had the stable genetic properties.The result of Dot-ELISA showed that the recombinant fowlpox virus expressed the hemagglutinin glycoprotein of AIV efficiently, and the expressing products possessed good antigenicity reacting with antibodies specifically. The result of Western-blotting demonstrated that hemagglutinin glycoprotein expressed by this recombinant virus existed with two cleavaged products of 44kDa and 23kDa representing HAl and L-1A2,there was no evidence of existence of HA0 with 63kDa.Sixty-day-old SPF chickens were immunized and challenged three weeks later. After challenged with 50 X LD50 of CDT isolate per chicken, all chickens vaccinated with the recombinant fowlpox virus and inactivated oil-emusion vaccine prepared with CDI isolate were survived, the control chickens and chickens immunized with fowlpox virus F-0l7 were died. The chickens vaccinated with recombinant fowlpox virus had HI antibody titer of 5.91og2 at 7 days postvaccination, and HI antibody titer rised to peak at 14 days postvaccination. HI antibody ~vas detaetable at 14 days postvaccination, and I-Il antibody titer increased to top at 35 days postvaccination in all chickens immunized with inactivated oilemusion vaccine. None I-Il antibody were detected in the control or fowlpox virus immunized chickens before or after immunization or challenge. Challenge virus was not reisolated from chickens vaccinated with the recombinant fowlpox virus and inactivated oil-emusion vaccine, and detected in everyone of all control or fowlpox virus immunized chickens.Virus was detected more frequently in long of control chickens.Determination of T lymphocytes indicated that vaccination of recombinant fowlpox virus and inactivated oil-emusion vaccine stimulated effectively cell-mediated immune response of chickens and activated I lymphocytes.The numbers of CD4-,CD8- and TCR 1-expressing cells was increased or did not changed in peripheral blood, thymus and spleen in chickens vaccinated with i~combinant fowlpox virus or inactivated oil-emttsion vaccine postchallenge, and decreased sharp in all control and fowlpox virus immunized chickens afler challenge.更多还原