猪轮状病毒和传染性胃肠炎病毒核酸免疫的研究
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摘要
本实验在MA104细胞上培养增殖了猪轮状病毒(RV)地方分离株JL94，并提取了病毒的双股RNA。以该RNA为模板，Li-9、Li-11为一对上、下游引物，Li-12、Li-13为另一对上、下游引物，通过RT-PCR分别扩增出了大小为1000bp、1300bp的两个片段。
将该片段插入T载体中构建了重组质粒pMD18-T-JL94/VP7和pMD18-T-JL94/VP6，经酶切初步鉴定和序列检测证明扩增的片段是JL94/VP7(1062bp)，JL94/VP6(1356bp)全长基因，并与国外参考毒株OSU株、Gottfried株进行了核苷酸和氨基酸序列的比较。JL94/VP7与OSU株VP7核苷酸同源性为99.9％，氨基酸同源性为99.9％，与Gottfried株VP7核苷酸同源性为74.1％，氨基酸同源性为75.4％。JL94/VP6与OSU株VP6核苷酸同源性为86.85％，氨基酸同源性为97.48％，与Gotffried株VP6核苷酸序列同源性为82.41％，氨基酸序列同源性为93.20％。
以pMD18-T-JL94/VP7为模板，Li-14、Li-11，为上、下游引物扩增带有Kozak序列的VP7基因并克隆到T载体上构建了重组质粒pMD18-T-VP7。将真核表达载体pcDNA3.1(+)与pMD18-T-VP7分别用HindⅢ、BamHⅠ双酶切，胶回收纯化后连接。经酶切鉴定和序列检测，证明VP7基因按设计要求已插入表达载体中，构建了重组真核表达载体pcDNA-VP7。
以重组质粒PHD为模板，Li-15、Li-2为上、下游引物扩增出上游带有BamHⅠ酶切位点的传染性胃肠炎病毒(TGEV)TH-98/N基因。将N基因胶回收纯化后与真核表达载体pcDNA3.1(+)分别用BamHⅠ、XhoⅠ双酶切，胶回收纯化后连接。经酶切鉴定和序列检测，证明N基因按设计要求已插入表达载体中，构建了重组真核表达载体pcDNA-N。
将昆明鼠随机分为A、B、C、D四组，分别肌肉注射pcDNA-VP7、pcDNA-N、pcDNA-VP7+pcDNA-N和pcDNA3.1(+)，每只鼠100μg/100μ1，共免疫3次，每次间隔2周。首次免疫第0d、14d、21d、28d、35d、39d、47d、54d采血分离血清用ELISA法检测抗体动态变化，同时检测外周血CD4~+、CD8~+T细胞动态变化。
A组小鼠血清在首免后第14d即可检出阳性抗体。以后在第28d、35d、39d、54d分别检出了阳性抗体(P/N≥2.0)，第35d抗体水平最高。C组小鼠血清在首免后第14d也可检出阳性抗体。以后在第28d、39d、54d均检出阳性抗体(P/N≥2.0)。B组小鼠在首次免疫后第39d检测到针对TGEVN蛋白的阳性抗体(P/N≥2.0)，其它时间均未检出阳性抗体。
A组与D组小鼠外周血CD4~+T细胞数量在首免后第14d、47d、54d有显著差异(P＜0.05)，A组小鼠外周血CD4~+T细胞数量明显高于D组。B组与D组小鼠外周血CD4~+T细胞数量在首免后第28d、47d分别出现显著差异(P＜0.05)。B组小鼠外周血CD4~+T细胞数量明显高于D组。C组与D组小员外周血CD4~+T细胞数量在首免后28d与对照组比较，差异显著(P＜0.05)，C组小员外周血CD4~+T细胞数量明显高于D组。
A组与D组小鼠外周血CD8~+T细胞数量在首免后第14d出现显著差异(P＜0.05)，
博士学位论文 猪轮状病毒和传染性胃肠炎病毒核酸免疫的研究 2002年 12月
A组小鼠外周血CDS”T细胞数量明显高于D组。B组与D组小鼠外周血CDS”T细胞
数量在首免后第14d出现显著差异（P＜0．0引，B组小鼠外周血CD矿T细胞数量明显高
于D组。C组与D组小鼠外周血CDS“T细胞数量在第35d出现显著差异（P＜0．05），C
组小鼠外周血CDS＋ T细胞数量明显高于D组。
本实验首次在国内克隆了猪轮状病毒地方分离株JL94的VP6、VP7全基因并与国
外参考毒株进行了序列比较分析，丰富了猪RV感染的分子流行病学资料，也为研制猪
轮状病毒基因工程疫苗提供了候选基因；首次在国内构建了猪轮状病毒地方分离株
JL94／VP7基因和传染性胃肠炎地方分离株h－98／N基因的重组真核表达载体
p。＊＊＊－VP 7和WDN AN：首次在国内应用重组真核表达载体WDN＊－W 7和WDN＊N
兔疫昆明鼠，结果证明上述重组真核表达载体能够诱导小鼠的体液免疫和细胞免疫应答。
该研究为今后对猪 RV和 TGEV进一步的分于生物学研究奠定了基础，为猪RV和
丁＊EV核酸疫苗的深入研究提供了科学的实验依据、理论资料以及技术和物质储备。
The native JL94 isolate of porcine rotavirus (RV) was propagated in MAl04 cel1 culture,
and harvested after 48 hours. Double-stranded RNA extracted from the virus was used as the
template for reverse transcription. Using two pairs of primers (Li-9 and Li-11, Li-12 and
Li-13), two gene segments were amplified by reverse transcription-polymerase chain reaction
(RT-PCR), respectively. The two gene segments were inserted into pMD18-T vector and
identified by sequencing, single and double digestion with restriction endonucleases,
respectively. One of the two gene segments was gene encoding oater capsid protein VP7,
another was gene encoding inter shell protein VP6. The two recombinant plasmids named
pMD18-T-JL94/VP7 and pMD18-T-JL94/VP6 were constructed. The result of their homology
comparison of nucleotide sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 74.1%,
respectively; with OSU/VP6, Gottfried/VP6 is 86.8%, 82.4%. The result of their homology
comparison of amino acid sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 75.4%, with
OSU/VP6, Gottfried/VP6 is 97.5%, 93.2%.
The gene of VP7 containing Kozak's sequence was amplified from pMD18-T-JL94/VP7
by PCR using a pair of prirner (Li-l4 and Li-11) designed according to the sequence of
JL94/VP7 gene cDNA, then it was inserted into pMD18-T vector and was identified by
digestion. The recombinant plasmid was pMD18-T-VP7. The VP7 gene from pMD18-T-VP7
digested by restriction endonucleases was recombinated into eukaryotic expression plasmid
pcDNA3.1(+). The recombinant plasmid, pcDNA-VP7, was constructed successfully after
being identified by sequencing and digestion.
The transmissible gastroenteritis virus (TGEV)TH98/N gene was amplified with a pair of
primer (Li-l5 and Li-2)from the recombinant plasmid, PHD. After being digested, it was
recombinated inio euknyotic expression plasmid pcDNA3. 1 (+). and named pcDNA-N.
The Kunming mice used for nucleic acid immtalzation were 6 to 8 weeks of age and
divided into four groups (35 mice each). Group A was injected with 100 μg of pcDNA-VP7,
group B with 100μg of pcDNA-N, group C with 100μg of pcDNA-VP7 and 100μg of
pcDNA-N, group D with 100μg of pcDNA3.1(+) in 100μg l volume via femoral quadriceps
intramuscular route, respectively, and the mice in ereh group were injected be times totally
After primary injection, all mice received booster injechon two thoes with the sazne dosage
and via the sarne route of primny injection at interval of two weeks. Blood saznples collected
3
from three mice at 0d before primary injection and those colIected from three mice of each
group at l4d, 2ld, 28d, 35d. 39d, 47d, 54d, were used for detecting antibody levels and the
changes of CD4+, CD8+ T cells in peripheral blood lyInphocytes.
The resultS showed that the anibodies against rotavirus JL94 couId be deteCted in the
serum of mice from grouP A at l4, 28, 35, 39 and 54 days of POstimmMon (PD, and the
same anibodies also could be detected in the serum of mice from group C at l4, 28, 39 and 54
days of PI. The anibodies to N protein of TGEV could be detected in the serum of mice frOm
group B at 39 days of PI.
There were significant differences rert0.05) in number of CD4+ T cells in Peripheral
blood lymphocytes between group A and group D at l4, 47 and 54 days of PI, group A was
higher than group D. There were sighficant differences (Prt0.05) in number of CD4+ T cel1s
in peripheral blood lymphocytes betWeen group B and group D at 28 and 47 days of PI, grOuP
B was higher than group D. There were significant differences (Pwt0.05) in number of CD4+ T
ceIls in peripheraI blood lymphocytes betWeen group C and group D at 28 days of PI, group C
was higher than grOup D.
There were significant differences (Prt0.05) in number of CD8+ T cells in Peripheral
bIood Iymphocytes betWeen group A and group D at l4 days of PI, group A was nigher than
group D. There were significant differences (P<0.05) in number of CD8+ T cells in Peripheral
b1ood lymphocytes b
将该片段插入T载体中构建了重组质粒pMD18-T-JL94/VP7和pMD18-T-JL94/VP6，经酶切初步鉴定和序列检测证明扩增的片段是JL94/VP7(1062bp)，JL94/VP6(1356bp)全长基因，并与国外参考毒株OSU株、Gottfried株进行了核苷酸和氨基酸序列的比较。JL94/VP7与OSU株VP7核苷酸同源性为99.9％，氨基酸同源性为99.9％，与Gottfried株VP7核苷酸同源性为74.1％，氨基酸同源性为75.4％。JL94/VP6与OSU株VP6核苷酸同源性为86.85％，氨基酸同源性为97.48％，与Gotffried株VP6核苷酸序列同源性为82.41％，氨基酸序列同源性为93.20％。
以pMD18-T-JL94/VP7为模板，Li-14、Li-11，为上、下游引物扩增带有Kozak序列的VP7基因并克隆到T载体上构建了重组质粒pMD18-T-VP7。将真核表达载体pcDNA3.1(+)与pMD18-T-VP7分别用HindⅢ、BamHⅠ双酶切，胶回收纯化后连接。经酶切鉴定和序列检测，证明VP7基因按设计要求已插入表达载体中，构建了重组真核表达载体pcDNA-VP7。
以重组质粒PHD为模板，Li-15、Li-2为上、下游引物扩增出上游带有BamHⅠ酶切位点的传染性胃肠炎病毒(TGEV)TH-98/N基因。将N基因胶回收纯化后与真核表达载体pcDNA3.1(+)分别用BamHⅠ、XhoⅠ双酶切，胶回收纯化后连接。经酶切鉴定和序列检测，证明N基因按设计要求已插入表达载体中，构建了重组真核表达载体pcDNA-N。
将昆明鼠随机分为A、B、C、D四组，分别肌肉注射pcDNA-VP7、pcDNA-N、pcDNA-VP7+pcDNA-N和pcDNA3.1(+)，每只鼠100μg/100μ1，共免疫3次，每次间隔2周。首次免疫第0d、14d、21d、28d、35d、39d、47d、54d采血分离血清用ELISA法检测抗体动态变化，同时检测外周血CD4~+、CD8~+T细胞动态变化。
A组小鼠血清在首免后第14d即可检出阳性抗体。以后在第28d、35d、39d、54d分别检出了阳性抗体(P/N≥2.0)，第35d抗体水平最高。C组小鼠血清在首免后第14d也可检出阳性抗体。以后在第28d、39d、54d均检出阳性抗体(P/N≥2.0)。B组小鼠在首次免疫后第39d检测到针对TGEVN蛋白的阳性抗体(P/N≥2.0)，其它时间均未检出阳性抗体。
A组与D组小鼠外周血CD4~+T细胞数量在首免后第14d、47d、54d有显著差异(P＜0.05)，A组小鼠外周血CD4~+T细胞数量明显高于D组。B组与D组小鼠外周血CD4~+T细胞数量在首免后第28d、47d分别出现显著差异(P＜0.05)。B组小鼠外周血CD4~+T细胞数量明显高于D组。C组与D组小员外周血CD4~+T细胞数量在首免后28d与对照组比较，差异显著(P＜0.05)，C组小员外周血CD4~+T细胞数量明显高于D组。
A组与D组小鼠外周血CD8~+T细胞数量在首免后第14d出现显著差异(P＜0.05)，
博士学位论文 猪轮状病毒和传染性胃肠炎病毒核酸免疫的研究 2002年 12月
A组小鼠外周血CDS”T细胞数量明显高于D组。B组与D组小鼠外周血CDS”T细胞
数量在首免后第14d出现显著差异（P＜0．0引，B组小鼠外周血CD矿T细胞数量明显高
于D组。C组与D组小鼠外周血CDS“T细胞数量在第35d出现显著差异（P＜0．05），C
组小鼠外周血CDS＋ T细胞数量明显高于D组。
本实验首次在国内克隆了猪轮状病毒地方分离株JL94的VP6、VP7全基因并与国
外参考毒株进行了序列比较分析，丰富了猪RV感染的分子流行病学资料，也为研制猪
轮状病毒基因工程疫苗提供了候选基因；首次在国内构建了猪轮状病毒地方分离株
JL94／VP7基因和传染性胃肠炎地方分离株h－98／N基因的重组真核表达载体
p。＊＊＊－VP 7和WDN AN：首次在国内应用重组真核表达载体WDN＊－W 7和WDN＊N
兔疫昆明鼠，结果证明上述重组真核表达载体能够诱导小鼠的体液免疫和细胞免疫应答。
该研究为今后对猪 RV和 TGEV进一步的分于生物学研究奠定了基础，为猪RV和
丁＊EV核酸疫苗的深入研究提供了科学的实验依据、理论资料以及技术和物质储备。
The native JL94 isolate of porcine rotavirus (RV) was propagated in MAl04 cel1 culture,
and harvested after 48 hours. Double-stranded RNA extracted from the virus was used as the
template for reverse transcription. Using two pairs of primers (Li-9 and Li-11, Li-12 and
Li-13), two gene segments were amplified by reverse transcription-polymerase chain reaction
(RT-PCR), respectively. The two gene segments were inserted into pMD18-T vector and
identified by sequencing, single and double digestion with restriction endonucleases,
respectively. One of the two gene segments was gene encoding oater capsid protein VP7,
another was gene encoding inter shell protein VP6. The two recombinant plasmids named
pMD18-T-JL94/VP7 and pMD18-T-JL94/VP6 were constructed. The result of their homology
comparison of nucleotide sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 74.1%,
respectively; with OSU/VP6, Gottfried/VP6 is 86.8%, 82.4%. The result of their homology
comparison of amino acid sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 75.4%, with
OSU/VP6, Gottfried/VP6 is 97.5%, 93.2%.
The gene of VP7 containing Kozak's sequence was amplified from pMD18-T-JL94/VP7
by PCR using a pair of prirner (Li-l4 and Li-11) designed according to the sequence of
JL94/VP7 gene cDNA, then it was inserted into pMD18-T vector and was identified by
digestion. The recombinant plasmid was pMD18-T-VP7. The VP7 gene from pMD18-T-VP7
digested by restriction endonucleases was recombinated into eukaryotic expression plasmid
pcDNA3.1(+). The recombinant plasmid, pcDNA-VP7, was constructed successfully after
being identified by sequencing and digestion.
The transmissible gastroenteritis virus (TGEV)TH98/N gene was amplified with a pair of
primer (Li-l5 and Li-2)from the recombinant plasmid, PHD. After being digested, it was
recombinated inio euknyotic expression plasmid pcDNA3. 1 (+). and named pcDNA-N.
The Kunming mice used for nucleic acid immtalzation were 6 to 8 weeks of age and
divided into four groups (35 mice each). Group A was injected with 100 μg of pcDNA-VP7,
group B with 100μg of pcDNA-N, group C with 100μg of pcDNA-VP7 and 100μg of
pcDNA-N, group D with 100μg of pcDNA3.1(+) in 100μg l volume via femoral quadriceps
intramuscular route, respectively, and the mice in ereh group were injected be times totally
After primary injection, all mice received booster injechon two thoes with the sazne dosage
and via the sarne route of primny injection at interval of two weeks. Blood saznples collected
3
from three mice at 0d before primary injection and those colIected from three mice of each
group at l4d, 2ld, 28d, 35d. 39d, 47d, 54d, were used for detecting antibody levels and the
changes of CD4+, CD8+ T cells in peripheral blood lyInphocytes.
The resultS showed that the anibodies against rotavirus JL94 couId be deteCted in the
serum of mice from grouP A at l4, 28, 35, 39 and 54 days of POstimmMon (PD, and the
same anibodies also could be detected in the serum of mice from group C at l4, 28, 39 and 54
days of PI. The anibodies to N protein of TGEV could be detected in the serum of mice frOm
group B at 39 days of PI.
There were significant differences rert0.05) in number of CD4+ T cells in Peripheral
blood lymphocytes between group A and group D at l4, 47 and 54 days of PI, group A was
higher than group D. There were sighficant differences (Prt0.05) in number of CD4+ T cel1s
in peripheral blood lymphocytes betWeen group B and group D at 28 and 47 days of PI, grOuP
B was higher than group D. There were significant differences (Pwt0.05) in number of CD4+ T
ceIls in peripheraI blood lymphocytes betWeen group C and group D at 28 days of PI, group C
was higher than grOup D.
There were significant differences (Prt0.05) in number of CD8+ T cells in Peripheral
bIood Iymphocytes betWeen group A and group D at l4 days of PI, group A was nigher than
group D. There were significant differences (P<0.05) in number of CD8+ T cells in Peripheral
b1ood lymphocytes b
引文
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