CN101603035A - Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing live vaccine - Google Patents

Abstract

The invention relates to a porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and a method for producing a live vaccine thereof. The GP2 gene of the classic porcine reproductive and respiratory syndrome virus (PRRSV) PTK strain is transformed by reverse genetic manipulation and gene recombination technology. After the second half of the GP3-GP5 gene, M protein gene, N protein gene and its spacer sequence and all nucleotide sequences of the 3' non-coding region were deleted, they were replaced with Chinese highly pathogenic porcine PRRS variant strains The corresponding gene of the chimeric recombinant vaccine is recombined into a chimeric recombinant vaccine strain (PC strain) containing the genes of the classic strain of porcine reproductive and respiratory syndrome and the variant strain at the same time, and the live vaccine produced by the chimeric recombinant vaccine PC strain has low toxicity and can be passed back The virulence of the fifth generation of pigs does not return to strong, and the genetic performance is stable; it has good immunogenicity and can protect the infection of classic strains of porcine PRRS and highly pathogenic variant strains at the same time.

Description

Porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain and method for producing live vaccine

technical field

The invention belongs to the technical field of veterinary biological products, and in particular relates to a method for constructing attenuated live vaccine strains by gene recombination technology and using the strains to produce live porcine reproductive and respiratory syndrome vaccines.

Background technique

Pig PRRS is a disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is clinically characterized by fever, abortion, increased mortality of piglets before and after weaning, and respiratory disorders in pigs of different ages. In 1991, Dutch scholar Terpstra et al. isolated European-type PRRSV from infected pigs and named it Lelystad virus (Lv); in the same year, American scholar Benfield et al. isolated American-type PRRSV and named it VR-2332. In recent years, Chinese scholars have successively isolated and identified strains such as CH-1a, S1, HB-1 and HB-2 of the American PRRSV.

Pig "high fever" is a major animal disease that has recently occurred in my country, and there has been no relevant research report at home and abroad before. Ning Yibao and others have confirmed through a large number of experiments that the infection of porcine PRRS virus is the main cause of high fever. And it was first reported in China.

There have been a lot of research results on the diagnosis and vaccine immunization of traditional pig PRRS at home and abroad, but these existing technologies cannot meet the needs of the prevention and control of highly pathogenic PRRS emerging in my country, and must be studied in a targeted manner Vaccine products and differential diagnosis techniques specifically targeting highly pathogenic porcine PRRS.

A large amount of experimental data proves that the pig PRRS live vaccine attenuated by artificial passage has the advantages of good immunity and long immunity period. common concern of the people. When using attenuated vaccines, due to the safety of the vaccine for pigs, it is generally only recommended to be used in areas where porcine PRRS has occurred, and it is not recommended to be used in farms where porcine PRRS has not occurred. The reason why the European Union prohibits the use of live vaccines to prevent pig PRRS is also because PRRS broke out after using attenuated live vaccines in some pig farms. The United States is also very cautious in the use of PRRS live vaccines. Although some other countries in the Americas and Asian countries do not object to the use of artificially passed attenuated PRRS live vaccines in areas where PRRS is endemic, they do not advocate the use of attenuated vaccines in pig farms where PRRS has not occurred. Inactivated vaccines are inactivated due to the inactivation of pathogens, so there is no problem of loosening the virus. Compared with live vaccines, the safety is better, but compared with live vaccines, the immune effect is poorer and the immunity period is shorter. The results of the use of highly pathogenic PRRS inactivated vaccines in my country have proved that in areas where PRRS has occurred, since the pig itself may have been infected with PRRS virus, the inactivated vaccine will not only fail to prevent The role of the disease, but will cause certain side effects. In terms of vaccine use targets, unlike live vaccines, inactivated vaccines are best used in areas where PRRS does not occur.

Different from the classic PRRS, highly pathogenic PRRS is caused by highly mutated strains of PRRS virus, neither the classic live vaccine nor the inactivated vaccine can prevent the occurrence of the disease. Therefore, in order to effectively prevent and control highly pathogenic PRRS, vaccine research is an urgent task. According to the experience and lessons of classic strain vaccine research, it is very necessary to carry out the research of new genetic engineering vaccine.

1. Routine vaccine against PRRS:

Along with the discovery of porcine PRRS, the vaccine research of porcine PRRS is just carried out thereupon, although everybody has mixed evaluation to the immune effect of existing porcine PRRS vaccine. At present, there are two types of porcine PRRS vaccines that have been commercialized at home and abroad: inactivated vaccines and attenuated live vaccines. There has also been a positive development trend in genetically engineered vaccine research.

1.1 Porcine PRRS attenuated live vaccine:

In terms of the classic attenuated live vaccine against PRRS, Boehringer Ingleheim of the United States first launched the commercial attenuated vaccine Resp PRRS/ReproTM in 1995, which has been approved for vaccination of pigs aged 3 to 18 weeks. Subsequently, several units have launched attenuated vaccines against porcine PRRS. At present, Spain, the Netherlands, the United States and China have developed and commercialized live attenuated PRRS vaccines. It can be used by pigs of all stages, but it is mainly used for inoculation of piglets aged 3 to 18 weeks to prevent and control respiratory disorders caused by PRRS. According to reports, animals can be completely protected when challenged with the homologous virulent virus 110 days after being inoculated with the attenuated PRRS vaccine, and the protection against the virulent challenge is even lifelong. The protective immune response of the pigs was stimulated 7 days after inoculation and lasted for more than 16 weeks. Compared with the unvaccinated control pigs, the shedding time of the vaccinated pigs after challenge was reduced, the lung lesions were alleviated, and the growth and development were good. However, most reports believe that there is a safety problem in the attenuated live vaccine of porcine PRRS. Botner et al. reported that in Denmark, more than 1,000 PRRS serologically negative pigs had used attenuated vaccines, but PRRS occurred in pigs soon after, and the vaccine virus was isolated from aborted fetuses and stillbirths, and the vaccine virus was found It can infect the fetus through the placenta and spread to unvaccinated sows, and some herds show acute PRRS-like syndrome. Some researchers also used the vaccine to vaccinate boars, and then challenged them with strong venom, and found that there were phenomena such as semen detoxification and decline in semen quality.

Opriessnig reported that a strain of PRRSV 98-38803 was isolated from a pig farm repeatedly inoculated with Ingelvac PRRS MLV, and it was confirmed that the strain came from Ingelvac PRRS MLV. Therefore, there is a possibility that the virulence of the attenuated PRRS vaccine will become stronger. From the above results, the attenuated PRRS vaccine has a fast immune production period, and is obviously better than the inactivated PRRS vaccine in controlling the clinical symptoms of PRRS. However, the reaction process of the virulent and attenuated PRRS virus in pigs is the same, so the safety problem caused by the long-term use of the attenuated PRRS vaccine is still the biggest controversy in pig PRRS immunization. Although attenuated vaccine immunization can make pigs obtain certain immunity, the unsafety of the attenuated vaccine, especially the possibility of variation such as virulence reversion, limits its wide use.

In terms of live attenuated vaccines against highly pathogenic PRRS, at present, although many units in my country are researching live attenuated vaccines against highly pathogenic PRRS through cell passage and attenuation, and have made good progress, their Safety needs further observation.

1.2 Inactivated vaccine against porcine PRRS:

In terms of the classic inactivated PRRS vaccine, Bayer launched the inactivated vaccine PRRomiSet M in 1997. Since then, Spain, the Netherlands, Canada and China have all developed inactivated PRRS vaccines and commercialized them. It is mainly used to prevent reproductive obstacles caused by gilts and multiparous sows infected with PRRS. The advantage of the inactivated vaccine is that it is safe, but it mainly stimulates the humoral immune response of the pig body, and it is powerless to clear the porcine PRRS virus in the infected macrophages, so the inactivated vaccine immunization alone cannot make the pig strong immunity . The disadvantages of inactivated vaccines after immunization are large immunization doses, many immunization times, and a long period of immunity generation, so they are not suitable for piglet immunization. According to reports, inactivated vaccine immunization can not protect piglets against porcine PRRS virus infection, and the duration of viremia and virus titer after virus infection are not significantly different from those of the non-immune group. Piglets and shelf pigs are the most important reason for the persistence of porcine PRRS virus in pig farms. Therefore, it is difficult to control and eradicate the existence and prevalence of porcine PRRS virus in pig farms through inactivated vaccines.

In terms of highly pathogenic PRRS inactivated vaccines, since the summer of 2006, porcine highly pathogenic PRRS outbreaks have occurred successively in some areas of my country. And other relevant units have carried out research on the highly pathogenic PRRS inactivated vaccine, which was officially put into production in May 2007. This vaccine uses the popular highly pathogenic PRRS mutant strain NVDC-JXA1 as the seed virus. This vaccine is currently the only inactivated vaccine that has obtained a provisional approval number and is used as an emergency preventive vaccine for government bidding and procurement. However, the results of use show that : This vaccine has the same problems as other inactivated vaccines.

At present, many units in my country are still researching live vaccines against highly pathogenic PRRS, but they are all under research and have not yet been put on the market.

2 Porcine Reproductive and Respiratory Syndrome Genetic Engineering Vaccine

There are many types of new PRRS vaccines currently being developed in the laboratory, mainly including subunit vaccines, genetically engineered recombinant/chimeric vaccines and DNA vaccines. Although the progress made is gratifying, they are basically in the research stage.

2.1 Subunit vaccines

At present, the research on the immunogenicity of each structural protein of porcine PRRS virus has been relatively clear. After porcine PRRS virus infects pigs, the immunoglobulins against porcine PRRS virus in serum are mainly aimed at N protein and M protein. Although GP3 cannot stimulate the production of neutralizing antibodies, it can protect piglets. GP4, GP5 and M protein can produce neutralizing antibodies, and GP5 has a stronger ability to produce neutralizing antibodies than GP4. Research on subunit vaccines and other new vaccines mostly targets these proteins.

2.2 Genetic engineering recombinant vaccine

Gagnon et al. carried out recombinant expression of genes such as GP5 and M protein, and constructed genetically engineered recombinant vaccines, which opened up a new field for the research of new vaccines against porcine PRRS. The work of artificially preparing recombinant viruses with porcine PRRS virus has also been carried out. Verheije et al. constructed the infectious cDNA clone of the LV strain of porcine PRRS virus, obtained artificially mutated porcine PRRS virus, and conducted a safety assessment of the mutant strains. and immunoprotective efficacy were tested. At present, genetically engineered recombinant vaccines can provide certain protection to pigs, but the effect is not complete.

2.3 DNA vaccine

Kwang, Pirzadeh, Yang Hanchun of China Agricultural University, etc. prepared DNA vaccines expressing porcine PRRS virus ORFK4, ORF5, ORF6 and ORF7 respectively and used them in animal experiments, showing that DNA vaccines can induce humoral immunity and cellular immunity.

Although there are many types of vaccines against PRRS, none of them can produce ideal safety protection for pigs.

The research and development of porcine reproductive and respiratory syndrome (or porcine PRRS) vaccine is a worldwide problem. Live vaccines generally have the problem of strong virulence, while the main problem of inactivated vaccines is poor immune efficacy. Currently, there is no satisfactory vaccine for the prevention of this disease in the world. And the highly pathogenic PRRS virus that appeared in my country in 2006, due to the mutation of the pathogenic gene, the classic PRRS vaccine basically has no immune protection to it. The inactivated vaccine produced can't produce effective protection to the wild strain equally, and there is the problem of virulence in the production of the attenuated vaccine of the attenuated strain after passage. Therefore, there is an urgent need to develop a safe and effective vaccine with a broad immune spectrum.

Contents of the invention

In order to overcome the problems and shortcomings of the above-mentioned prior art, the present invention provides a chimeric recombinant vaccine strain of porcine reproductive and respiratory syndrome virus and a method for producing live vaccines thereof. The gene of the diseased porcine reproductive and respiratory syndrome virus strain is chimerized with the gene of the classical porcine reproductive and respiratory syndrome attenuated strain, and recombined into a new chimeric attenuated vaccine strain and used to produce vaccines, which effectively solves the problem of the existence of such vaccines. above question.

Technical scheme of the present invention is:

Porcine Reproductive and Respiratory Syndrome Virus Chimeric Recombinant Vaccine Strain, Using Reverse Genetic Operation and Gene Recombination Technology to Introduce the Second Half of GP2 Gene, GP3-GP5 Gene, After all the nucleotide sequences of the M protein gene, the N protein gene and their spacer sequences and the 3' non-coding region are deleted, they are replaced with the corresponding genes of the highly pathogenic porcine PRRS variant strain in China, and recombined into a porcine The chimeric recombinant vaccine strain (PC strain) of reproductive and respiratory syndrome classic strain and variant strain gene, the total length of the gene sequence of the chimeric recombinant vaccine strain (PC strain) is 15562bp, the chimeric recombinant vaccine strain (PC strain) gene amino acid sequence. The chimeric recombinant strain has low toxicity, is safe for pigs and has good immunogenicity, and the PC strain can be used to produce vaccines to prevent infection of porcine reproductive and respiratory syndrome classic strains and variant strains.

The corresponding genes of the highly pathogenic porcine blue ear disease variant strain in China are the second half of the GP2 gene, GP3 gene, GP4 gene, GP5 gene, M protein gene, The N protein gene and its spacer sequence and all nucleotide sequences of the 3' non-coding region, and the total length of the embedded target nucleic acid sequence is 2421bp, mainly encoding the structural protein of PRRSV and the 3' non-coding region. The second half of the GP2 gene, GP3 gene, GP4 gene, GP5 gene, M protein gene, and N protein gene are structural proteins of the virus, and the nucleotide sequence of the 3' non-coding region is related to the replication and activity of the virus. It can be verified by amplification by specific RT-PCR method. Amino acid sequence of the corresponding gene of the embedded highly pathogenic porcine PRRS variant strain in China.

The method for producing the live vaccine by the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain is carried out in the following steps:

(1) Passage and culture of cells for seedling production: Digest and pass on Marc145 or MA104 cell lines with EDTA-trypsin cell dispersion, continue to culture with cell growth medium, and form a dense monolayer for later use; for continued passaging or inoculation Virus;

(2) Propagation of cytotoxic species: Add the PC strain virus solution to the cell bottle that has grown into a good cell monolayer according to the volume of 1/10, after 1 hour of adsorption, add the cell maintenance solution to the cell line monolayer, and continue Cultivate, harvest when 70-80% of the cells have lesions, freeze and thaw the harvested venom 2-3 times and store it below -15°C, take a small amount for inspection of semi-finished products;

(3) Mixing seedlings, sub-packaging and freeze-drying: Mix the qualified virus culture solution and conventional stabilizer in a volume ratio of 1:1 in a container, shake well, and quantitatively sub-package; each head contains cytovenom Not less than 10 ^5.0 TCID ₅₀ , and freeze-dry quickly after subpackaging to obtain the finished product.

The porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC strain of the present invention is the first half of the ORF1-ORF2 gene fragment of the classical virus PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV) as a carrier, which is inserted into the highly pathogenic pig reproductive and respiratory syndrome virus. The second half of the ORF3-ORF7 gene fragment of the ORF2 gene fragment of the respiratory syndrome strain isolated in China was constructed into a recombinant chimeric PC strain, and the vector name was PB13117 plasmid. The nucleic acid sequence of the vector includes: PBluescript SK complete sequence, CMV promoter, T7 promoter and the genome sequence of porcine reproductive respiratory syndrome virus (PRRSV) PTK strain, the size of the CMV promoter T7 promoter of PBS13117 plasmid is respectively 565bp and 19bp. Only the T7 promoter is used in the present invention, the terminator is the t1t2 tandem transcription terminator of the rrnB rRNA operon, the size is 402bp, the CMV promoter comes from cytomegalovirus, the T7 promoter comes from bacteriophage, and the rrnB terminator comes from Escherichia coli, after inserting the target nucleic acid sequence of China's highly pathogenic PRRS variant strain, the complete gene sequence of the chimeric vaccine virus PC strain is 15562bp. The technological process for constructing the chimeric recombinant PC strain of the present invention is shown in FIG. 1 .

The marker gene is ampicillin resistance gene (Ampr), the ORF size of the gene is 861bp, its function is to enable bacteria containing the gene to have the ability to tolerate a certain concentration of ampicillin, and it is derived from Escherichia coli.

Gene manipulation method:

The gene manipulation methods used in the present invention include conventional molecular biology methods such as RT-PCR, enzyme digestion, connection, transformation, in vitro transcription, and cell transfection.

The method for detecting the transgenic microorganisms of the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC strain adopts RT-PCR method for detection.

The present invention has the following advantages:

Proliferation and genetic stability of chimeric recombinant PC strains on cells: After successful virus rescue, the chimeric recombinant PC strains of the present invention can be stably expressed and function in Marc145 or MA104 cells, and the constructed chimeric strains can be Marc145 or MA104 cells can proliferate stably, and their various characteristics have not changed after continuous passage for more than 20 generations. In this animal body, the virulence has not returned to strong after five generations, and the genetic performance is stable.

Vaccine production and vaccine characteristics: The chimeric recombinant PC strain of the present invention has good safety and immunogenicity to the animal, so it is used to produce vaccines to prevent porcine reproductive and respiratory syndrome classic strains and highly pathogenic variants strain infection.

The chimeric recombinant PC strain constructed by the present invention can proliferate efficiently on Marc145 or MA104 cells, causing them to produce pathological changes. The strain has low virulence, and the virulence of back-transferred pigs does not return to strong in the 5th generation, and the genetic performance is stable; The gene structure of the pathogenic mutant strain, the vaccine made with it has high immune efficacy, and can simultaneously protect the infection of the classical strain of porcine PRRS and the highly pathogenic mutant strain.

The vaccine produced by the present invention has the following advantages:

1. Due to the clear genetic background, the vaccine will not appear to become more virulent, which can overcome the problem of artificial passaging to weaken the safety of the vaccine.

2. Because it is a live vaccine, it can stimulate both humoral immunity and cellular immunity, and the gene is not affected by the mutagenesis of long-term passage, and the gene structure is closer to the original strain. Should have a good immune effect.

3. Since the vaccine contains both some genes of the classic strain and some genes of the mutant strain, theoretically speaking, it is a bivalent vaccine, which can be used to prevent both the classic PRRS and the highly pathogenic Sexual swine blue ear disease.

4. Since the expression gene can be artificially inserted in the gene construction, the vaccine can be used for differential diagnosis. This is very beneficial for evaluating the immune efficacy of vaccines and eradicating diseases, which is difficult for traditional vaccines.

5. Compared with the passage of the virus in cells for a long time to weaken it, the time spent on the construction of genetically engineered vaccines is much shorter, especially under the premise that we have obtained a safe vector. This is particularly meaningful when my country is in urgent need of a highly pathogenic PRRS vaccine.

Description of drawings

Fig. 1 is a flow chart of the process for constructing a chimeric recombinant PC strain according to the present invention.

Detailed ways

In the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain of the present invention, the second half of the GP2 gene and the GP3～ After the GP5 gene, M protein gene, N protein gene and its spacer sequence and all nucleotide sequences of the 3' non-coding region are deleted, they are replaced with the corresponding genes of the highly pathogenic porcine PRRS variant strain in China, and recombined into a A chimeric recombinant vaccine strain (PC strain) containing porcine reproductive and respiratory syndrome classic strain and variant strain genes at the same time, the corresponding gene of the Chinese highly pathogenic porcine blue ear disease variant strain is Chinese highly pathogenic pig indigo All nucleotide sequences of the second half of the GP2 gene, GP3 gene, GP4 gene, GP5 gene, M protein gene, N protein gene and their spacer sequences and 3' non-coding regions of the ear disease mutant strain, and the inserted target nucleic acid The total length of the sequence is 2421bp, mainly encoding structural proteins and 3' non-coding regions of PRRSV.

The method for producing the live vaccine by the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain is carried out in the following steps:

(1) Passage and culture of cells for seedling production: Digest and pass on the Marc145 cell line with EDTA-trypsin cell dispersion liquid, continue to culture with cell growth medium, and when a good monolayer is formed, use it for standby; for continued passaging or virus inoculation;

(2) Propagation of cytotoxic species: Add the PC strain virus solution to the cell bottle that has grown into a good cell monolayer according to the volume of 1/10, after 1 hour of adsorption, add the cell maintenance solution to the cell line monolayer, and continue Cultivate, harvest when 70-80% of the cells have lesions, freeze and thaw the harvested venom 2-3 times and store it below -15°C, take a small amount for inspection of semi-finished products;

(3) Mixing seedlings, subpackaging and freeze-drying: Mix the qualified virus culture solution with a conventional stabilizer in a volume ratio of 1:1 in a container, shake well, and quantitatively subpackage; each portion contains cells The venom is not less than 10 ^5.0 TCID ₅₀ , and the finished product is obtained by quickly freeze-drying after subpackaging.

The characteristics of the vaccine strain produced by the present invention on cells: using the PB13117 plasmid as a carrier, the chimeric vaccine virus PC obtained after inserting the second half of ORF2 and the nucleic acid sequence of ORF3-ORF7 of the highly pathogenic porcine PRRS virus GD strain in China strain, which can stably proliferate in Marc145 cells, causing the cells to develop lesions in the form of a stretch net in about 72 hours. After continuous passage on the cells for 20 generations, the proliferation characteristics of the virus have not changed, and the gene structure has not changed.

1. The safety of the vaccine for this animal:

1. Recombinant porcine PRRS vaccine (4th generation) (TCID ₅₀ =10 ^5.25 ) was used to infect 10 healthy piglets at the age of 33 days respectively, the infection dose was 3ml/pig, 2ml nasal drip, 1ml intramuscular injection in the neck, after infection The body temperature was measured once a day in the morning and afternoon, and the clinical symptoms were observed. After 30 days, 5 pigs were slaughtered and dissected to observe the changes of their lungs and other organs.

Results All 10 pigs had no body temperature rise, and all pigs lived healthy. All internal organs of the 5 pigs dissected showed no visible pathological changes.

2. Infect respectively 5 healthy piglets at the age of 35 days with pig PRRS recombinant vaccine (the 5th generation) (TCID ₅₀ =10 ^5.0 ), the infection dose is 3ml/pig, 2ml nasal drip, 1ml neck intramuscular injection, infection After that, the body temperature was measured once a day in the morning and afternoon, and the clinical symptoms were observed. After 21 days, all the animals were slaughtered and dissected to observe the changes of their lungs and other organs.

Results All five pigs had no body temperature rise, and all pigs lived healthy. All internal organs of 5 pigs dissected showed no visible pathological changes

3. The ability to strengthen the virulence.

The PRRSV PC strain was continuously passed back in pigs for 5 generations, and no virulence reversion phenomenon was found.

The results showed that the recombinant PRRS vaccine was safe for pigs.

2. Efficacy test of PC strain vaccine against PRRS:

1. Immunize 15 healthy piglets with recombinant PRRS vaccine (PC strain 4th generation) (TCID ₅₀ =10 ^5.25 ), challenge the virus 35 days later, observe for 21 days after the challenge, dissect and observe the lungs and other organs after slaughter The change. The test results are as follows:

Safety: All immunized pigs had no temperature rise within 35 days after vaccination. There were no clinical symptoms. Prove that vaccines are safe.

Immune protection: 100% positive in antibody detection 14 days after immunization; all immunized pigs were healthy and alive after challenge with virulence, and the weight gain of immunized pigs in three groups was significantly higher than that of control pigs. The lung lesion protection rate of immunized pigs was 78%.

2. Use two groups of PRRS recombinant vaccines (PC strain 5th generation) (TCID ₅₀ =10 ^5.25 ), after 5-fold dilution, immunize 5 36-day-old healthy piglets in each batch, and intramuscularly inject 1ml/head into the neck 21 days later, together with the control 5 heads, each head nods 0.5ml and injects 2ml into the neck muscle. The temperature was measured every day, the clinical symptoms were observed, and the changes in the lungs and other organs were dissected after 21 days of slaughter. The result is as follows.

In one group of immunized pigs, 2/5 got sick and 1/5 died, in the other group 1/5 got sick and 0/5 died, and in the control pigs 5/5 got sick and 4/5 died.

The results showed that immunizing pigs with the recombinant PRRS vaccine had a good protective effect.

attachment1:

CTAGTG A TTACG CCTGGCTGGCGTTCCTGTCCTTCAGCTACACGGCCCAGTTCCATCCCGAGATATTGGGATAGGGAATGTGA

GTCAAGTTTTATGTTGACATCAAGCACCAATTCATCTGCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCATGACAATA

TTTCAGCCGTA TT TCAGACCTACTACCAACACCAGGTCGACGGCGGCA A TTGGTTTCACCTGG A ATGGCTGCGTCCTTTCTTTT

CCTCTTGGTTGGTTTTAAATGTTTCGTGGTTTCTCAGGCGTTCGCCTGCAAGCCATGTTTCAGTTCGAGTCTTTCGGACATCAA

AACCAACACCACCGCAGCATCAGACTTCGTTGTCCTCCAGGACATCAGCTGCCTTAGGCATGGCGACTCGTCCTCTCCGACGAT

TCGCAAAAATTTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGTACATCACCATCACTGCCAATGTCACAGATGAAAATTATCT

ACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCCGAGATGAGTGAAAAGGGATTCAAAGTGGTGTTTGG

CAATGTGTCAGGCACTGTGGCTGTGTGCGTCAACTTTACCAGCTACGTCCAACATGTCCAAGGAGTTTACCCAACGCTCCTTAGT

GGTCGATCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGAGGTGGGCAACCGTTTTTAGCCTGTCTTTTTGCCATCCT

ACTGGCAATTTGAATGTTCAAGTATGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTGCTTTTTTTGTGGTGTATCG

TGCCGTTCTATCTTGCTGTGCTCGTCAACGCCAGCAACAACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGTTATGTG

AGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGGAGACTTTTGTCATTCTTCCCCGTGTTGACTCACATTG

TTTCCTATGGGGCACTCACCAGCCATTTCCTTGACACAGTTGGTTTGGCCACTGTGTCTACCGCCGGATATTATCACGGGC

GGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGT

CCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTG

TGGAGAAAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTT

TAACCAGAGTTTCCAGCGGAACGATGGGGTCGTCTCTAGACGACTTCTGCAATGATAGCACAGTCCACAGAAGGTGCTTTTGGC

GTTTTCCATTACCTACACGCCAGTGATGATATATGCTCTAAAGGTAAGTCGCGGCCGACTGCTAGGGCTTCTGCACCTTTTGAT

CTTTCTGAATTGTGCTTTTACCTTCGGGTACATGACATTCGTGCACTTTGAGAGCACAAATAGGGTCGCGCTCACTATGGGAGC

AGTAGTTGCACTTCTTTGGGGAGTGTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCAGATGCCGTTTGTGCTTGCTAGG

CCGCAAGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATCCGATTGCGGCAAATGATAACCACGCATT

TGTCGTCCGGCGTCCCGGTCCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGC

TGTTAAGCAGGGAGTGGTAAACCTTGTTAAATATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGC

CAGTCAATCAGCTGTGCCAAATGCTGGGTAAGATCATCGCCCAACAAAACCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATA

GGAAGAAAAACCCGGAGAAGCCCCATTTTCCCTCTAGCGACTGAAGATGACGTCAGGCATCACTTTTACCCCTAGTGAGCGGCAAT

TGTGTCTGTCGTCGATCCAGACTGCCTTCAATCAGGGCGCTGGAACTTGTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTG

TGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCAGCATGATGGGCTGGCATTCTTT

GGCACCTCAGTGTTAGAATTGGGAGAATGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTCACCTATTCAATTAGGG

CGACCGTGTGGGGGTAAAGTTTAATTGGCGAGAACCATGCGGCCGTAATTAAAAAAAAAAAAAAAAAAA

Attachment 2:

TATGACGTATAGGTGTTGGCTCTATGCCTTGACATTTGTATTGTCAGGAGCTGTGATCATTGACACAGCCCAAAGCTTGCTGCACAGAAACACCCTTC

TGTGACAGCCTCCTTCAGGGGAGTTTAGGGGTCGTCCCTAGCACCTTGCTTCCGGAGTTGCACTGCTTTACGGTCTCTCACCCCTTTAACCATGTCT

G G GATACTTGATCGGTGC A CGTGTACCCCCAATGCCAGGGTGTTCATGGCGGAGGGCCAAG T CTACTGCACACGATG CC TCAGTGCACGGTCTCTCCT

TCCTCTGAATCTCCAAAGTTCCTGAACTCGGGGTGCTGGGCCTATTCTATAGGCCCGAAGAGCCACTCCGGTGGACGTTGCCACGTGCATTCCCCACTG

TTGAGTGCTCCCCCGCCGGAGCCTGCTGGCTTTCTGCCATCTTTCCAATTGCACGAATGACCAGTGGAAATCTGAACTTTCAACAAGAATGGTGCGG

GTCGCAGCTGAGCTTTACAGAGCCGGCCAGCTCACCCCTGCAGTCTTGAAGACTCTACAAGTTTATGAACGGGGTTGCCACTGGTACCCCATTGTTGG

ACCTGTTCCTGGAGTGGCCGTTTATGCCAACTCCCTACATGTGAGTGATAAACCTTTCCCGGGAGCAACTCACGTGTTAACCAACCTGCCGCTCCCGC

AGAGACCCCAAGCCTGATGATTTTTGCCCCTTTGAGTGTGCTATGGCTACTGTCTATGACATTGGTCATGACGCCGTCATGTATGTGGCCGCAGAGAAA

GTCTCCTGGGCCCCTCGTGGCGGGGATGAAGTGAAATTCGAACCTGTCCCCGGGGAGTTGAAGTTGATTGCGAACCGACTCCGCACCTCCTTCCCGCC

CCACCACGCAGTGGACATGTCTAAGTTCACCTTCACAGCCCCTGGGCGTGGTGTTTCTATGCGGGTCGAACGCCAACACGGCTGCCTCCCCGCTGACA

CAGTTCCTGAAGGCAACTGCTGGTGGAGCTTGTTCAACTTGCTCCCACTGGAAGTTCAGAACAAAGAAATTCGTCATGCCGGCCAATTTGGCTACCAG

ACTAAGCATGGTGTCTCTGGCAAGTACCTACAGCGGAGGCTGCAAGTTAATGGTCTTCGAGCAGTAACTGACCTAAATGGACCTATCGTCGTACAGTG

CTTCTCCGTTAACGAGAGTTGGATCCGCCACTTGAAACTGGCGGAAGAACCCAGCTACCCTGGGTTTGAGGACCTCCTCAGAATAAGGGTTGAGCCCA

ACACGTCGCCATTGGCTGACAAGGATGAAAAAATTTTCCGGTTTGGCAATCACAAGTGGTATGGCGCTGGAAAGAGAGCAAGGAAAGCACGCTCTAGT

GCGACTGCTACAGTCGCTGGCCGCGCTTTGCCCGTTCGTGAAACCCGGCAGGTCGAGGAGCACGAGGTTGCCGGCGCCAACAAGGCTGAGCACCTCAA

ACACTACTCCCCGCCTGCCGAAGGGAATTGTGGTTGGCACTGCATTTCCGCCATCGGCAACCGGATGTTGAATTCCAAATTTGAAACCACCCTTCCCG

AAAGAGTGAGACCTCCAGATGACTGGGCTACTGATGAGGATCTTGTGAATGCCATCCAAATCCTCAGACTCCCTGCGGCCTTGGACAGGAACGGTGCT

TGTGCTAGCGCCAAGTACGTACTTAAGCTGGAAGGTGAGCATTGGACTGTCACTGTGACCCCTGGGATGTCCCTTCTTTGCTCCCTCTTGAATGTGT

TCAGGGCTGTTGCGAGCATAAGGGCGGTCTTGGTTCCCCAGATGCAGTCGAGGTTTTCGGATTTGACCCTGCCTGCCTTGACTGGCTGGCTGAGGTGA

TGCACTTGCCTAGCAATGCCATCCCAGCCGCTCTGGCCGAAATGTCCGGCGATTCCAATCGTCCGGCTTCCCCGGTCACCACCGTGTGGACTGTTTCG

CAGTTCTTAGCCCGCCACAACGGAGGGAATCACCCTGACCAAATACGCTTAGGGAAAATTATCAGCCTTTGTCAGGTGATTGAGGACTGCTGCTGTTC

CCAGAACAAAACCAACCGGGTCACCCCGGAGGAGGTCGCAGCAAAGATTGACCTGTACCTCCGTGGTGCAACAAATCTTGAAGAATGCTTGGCCAGGC

TTGAGAAAGCGCGCCCGCCACGCGTAATGGACACCTCCTTTGATTGGGATGTTGTGCTCCCTGGGGTTGAGGCGGCAACTCAAACGACCGAACTGCCC

CAAGTCAACCAGTGTCGCGCTCTGGTCCCTGTTGTGACTCAAAAGTCCTTGGACAACAACTCGGTCCCCTCTGACCGCCTTTTCACTGGCTAACTACTA

CTACCGCGCGCAAGGTGACGAAGTTCGTCACCGTGAAAGACTAACCACCGTGCTCTCTAAGTTGGAAGGGGTTGTTCGAGAAGAATACGGGCTCATGC

CAACCGGGCCTGGTCCACGGCCCACACTGCCACGCGGGCTCGACGAACTCAAGGACCAGATGGAGGTGGACTTGCTGAAACTGGCTAACGCCCAGATG

ACTTCGGACATGATGGCCTGGGCAGTCGAGCAGGTTGACCTAAAGACTTGGGTCAAGAACTATCCGCGGTGGACACCACCACCTCCTCCGCCAATAGT

TCAGCCTCGAAAAACGAAGCTTGTCAAGAGCTTACCAGAGAGCAAGCCTGTTCCTGCACCGCGTAGGAAGGTCAGGTCCGATTGTGACTGCCCCACCC

TATCGGGCAACAATCTTCCTGACAGTTGGGAAGATTTGGCTGTTGGTTGCCCCTCTGATCTCCCTACCTCACCTGAGCCGGTAACACCTTTGAGTGAG

CCGGCATCTGTGTCCGCACCGCGACGCTCTTTTAGGCCGGTGAAGCCTTTGAGTGAACCAGTTCCAGTCCCTGCACCGCGCAAGACTGTGTCCCGACC

GGCAACACCTCTGAGTGAGCCGATCCCTGTGCCCGCACCGCGACGCAAGTTTCAGCAGGTAGAAAAAGTGAATCCGGCGGCGGCAACCCTGGCGTGCC

AAGACGAGTTTCCAGATTTGTCTGCATCCTCGCATACTGAATATGAGGCGTCTCCCCCTTGTACTACCGCAGAACGGGGACGTTCTTGAAGTGGAGGAG

CGGGAAGCTGAGGAAATCCTGAGTGGAATCTCAGACATACTGGATGCCATCAAACCGGCATCTGCATCATCAAGCAGCTCCCTGTCAAGTGTGGCGAT

CACACGCCCGAAATACTCAGCTCAAGCCATCATTGACTCGGGTGGGCCCTACAGCGGGCATCTCCAAGAGGTGAAGGAAACATGCCTAAGCATCATGA

GTGAGGCATGTGATGTGACCAAGCTTGATGACCCTGCCACGCAGGAATGGCTTTCTCGCATGTGGGATAGGGTGGACATGCTGACTTGGCGCAATACG

TCTGTTCACCAGGCGTCTCGCACCTTGGACGACAGATTTAAGTTTCTCCCGAAGATGATACTTGAAACACCGCCGCCCTACCCGTGTGGGTTCGTGAT

GATGCCTCGCACACCTGCACCCTCCGTGGGTGCGGAGAGCGACCTCACTATTGGCTCAGTCGCTACTGAGGGCGTTCCACGCATCTTCGGGAAAGTAA

ATGATGTCTGCAAGATGATCGACCAGAGACCCTTGGTACTCTTTGAAAATGAGCTGGCAGATGACCAACCTGCCAGAGATCCTCGGACATCATCGCAG

AGGTTTGACGGGAGCACACCAGCTCCGCCCGCAGGCACGGATGGCACCGGTTTGGCTTCGGGCCCTGGAGTGAGAGAAGTGGATTCATGTGAGGCGAG

CTCAACCGAGAAAAATGAACAGCCCTTCGTGTTGAACGGCGGCGCCAGCACACAGGCGTCAACGTTTACCAATTTGCCGCCTCCAGGCGGTATAGATG

CGGGCGGGAGTGGGCCGTTACAAACGGTGCGAAAGAAGGCTGAACGGTTCTTTGACCTACTAAGCCGTCAGGTTTTTAATCTCGTCTCCCATCTCCCT

GTTTTCTTCTCACGCCTTTTCAAACCTGGCGGTGACTATTCTCCGGGTGATTGGGGTTTTGCAGCTTTTACTTTATTGTGCCTCTTTTTGTGTTACAG

TTACCCGGCCTTTGGTGCTGTTCCCCTTGGGTGTATTTTCTGGGTCTTCTCGGCGTGTTCGAATGGGGGTTTTTGGCTGCTGGTTGGCTTTTGCTG

TTAGTCTGTTCAAACCTGTGTCCGACCCAGTCGGCGCTGCTTGTGAATTTGATTCGCCAGAGTGTAGAAACATCCTTCATTCTTTTGAGCTTTCCAAA

CCTTGGGACCCTGTTCGCGGCCTTGTTGTGGGCCCCGTCGGTCTCAGTCTTGCCATTTTTGGCAGGTTATTGGGCGGGGCACGCCACATCTGGCACTT

TTTGCTTAGGTTTGGCATTGTTGCAGATTGTATCTTGGCTGGAGCTTATGTGCTTTCTCAAGGCAGGTGTAAAAAGTGCTGGGGATCTTGTATAAGAA

CTGCTC C TAATGAGGTCGCCTT TA ACGTGTTTCTTTTACACGTGCGACCAGGTCGTCACTTATC GA CCTGTGCAATCGGTT TT GCGCGCCAAAAGGT

ATGGACCCCATTTTCCTCGCCACTGGGTGGCGCGGGTGCTGGACCGGCCGAAGCCCCATTGAGCAACCCTCTGAAAAACCCATCGCGTTTGCCCAGTT

GGATGAAAAAAAGATTACGGCTAGGACTGTGGTCGCCCAGCCCTATGACCCCAACCAAGCTGTAAAGTGCTTGCGGGTATTGCAGGCGGGCGGGGTGA

TGGTGGCTGAGGCAGTCCCAAAAGTGGTCAAAGTTTCTGCTGTTCCATTCCGAGCCCCCTTCTTTCCCACCGGAGTGAAAGTTGATCCTGAATGCAGG

ATTGTGGTTGACCCCGACACTTTCACTGCAGCCCTCCGATCTGGCTACTCCACCACAAACCTCGTCCTTGGTGTGGGGGACTTTGCCCAGCTGAATGG

ATTAAAAATCAGGCAAATTTCCAAGCCTTCAGGGGGAGGTCCACACCTCATGGCTGCCCTGCATGTTGCTTGCTCTATGGCTCTGCACATGCTTGTTG

GGATTTATGTGACTGCTGTGGGTTCTTGCGGCACCGGCACTAACGATCCGTGGTGCGCCAACCCGTTTGCCGTCCCTGGCTACGGACCTGGCTCTCTC

TGCACGTCCAGATTGTGCATCTCCCAACATGGCCTTACCCTACCCTTGACAGCACTTGTGGCGGGATTCGGCTTTCAGGAAATTGCCTTGGTTATTTT

GATTTTTGTTTCTATCGGAGGCATGGCTCATAGATTGAGTTGCAAGGCTGATATGCTGTGTATTTTGTTTGCAATCGCCAGCTATGTTTGGGTACCTC

TTACCTGGTTGCTTTGTGTGTTTCCTTGCTGGTTGCGCTGTTTTTCGTTGCACCCCCTCACCATCCTATGGTTGGTGTTTTTCCTGATTTCTGTAAAT

ATGCCTTCAGGAATCTTGGCCTTGGTGTTGTTGATTTCCTCTGGCTTCTTGGTCGTTATACCAACGTTGCCGGTCTTGTCACTCCCTATGACATTCA

TCATTACACCAGTGGCCCCCGCGGTGTTGCCGCCTTGGCTACCGCACCAGATGGGACCTACTTGGCCGCTGTCCGCCGCGCCGCGTTGACTGGTCGCA

CCATGCTGTTTACCCCGTCTCACGCTCGGGTCCCTTCTTGAGGGCGCTTTTCAGAACTCGAAAGCCCTCACTGAACACCGTTAATGTGGTCGGGTCCTCC

ATGGGCTCTGGCGGGGTGTTCACCATCGACGGGAAAATTAAGTGCGTAACTGCTGCACATGTCCTTACGGGTAATTCAGCTAGGGTTTCCGGGGTTGG

CTTCAATCAAATGCTTGACTTCGATGTGAAAGGAGACTTCGCCATAGCCGATTGCCCAGACTGGCAAGGGGCTGCTCCCAAGACCCAATTCTGCGAGG

AAGGATGGACTGGCCGGGCCTATTGGCTAACGTCTTCTGGTGTCGAACCCGGCGTCATTGGAAAAGGATTCGCCTTCTGCTTCACCGCGTGCGGCGAT

TCCGGATCCCCAGTAATCACCGAGGCCGGCGAGCTTATCGGCGTTCACACGGGGTCAAATAAACAAGGAGGAGGCATCGTCACGCGCCCCTCAGGCCA

GTTTTGTAGTGTGGCACCCGTCAAATTAAGCGAACTAAGTGAATTCTTTGCAGGGCCTAAGGTCCCGCTCGGTGATGTGAAAGTTGGCAGCCACATAA

TTGAAGACGTAGGCGAGGTGCCTTCAGATCTTTGCGCCTTGCTTGCTGCCAAACCTGAACTGGAAGGAGGCCTCTCCACCGTTCAACTTCTGTGTGTG

TTTTTCCTCCTGTGGAGAATGATGGGACATGCCTGGACGCCCTTGGTTGCCGTAGGGTTTTTTTATCTTGAATGAGGTCCTCCCAGCTGTCCTGGTCCG

GAGTGTTTTCTCCTTTGGAATGTTTGTGCTATCCTGGCTCACACCATGGTCTGCGCAAGTTCTGATGATCAGGCTTCTAACAGCAGCTCTTAACAGGA

ATAGATGGTCACTTGCCTTTTACAGCCTCGGTGCAATGACTGGTTTTGTCGCAGATCTCGCGGCTACTCAGGGGTATCCGTTGCAGGCAGTGATGAGT

TTGAGCACTTATGCATTCCTGCCTCGGATAATGGTTGTGACTTCACCAGTCCCAGTGGTTGCGTGTGGTGTTGTGCACCTACTTGCCATCATTTTGTA

CTTGTTTAAGTACCGCTGCCTGCACAACATCCTTGTTGGCGATGGAGTGTTCTCTGCGGCTTTCTTCCTGCGATATTTTGCCGAGGGAAAGTTGAGGG

AGGGGGTGTCGCAATCCTGCGGGATGAATCATGAGTCACTTACCGGTGCCCTCGCTATGAGACTCAATGACGAGGACTTGGATTTCCTCACGAAATGG

ACTGATTTCAAGTGCTTTGTTTCTGCGTCCAACATGAGAAATGCTGCGGGCCAATTCATCGAGGCTGCCTATGCTAAAGCACTTAGAGTAGAACTTGC

CCAGTTGGTGCAGGTTGATAAGGTTCGGGGTACTTTGGCCAAACTTGAAGCTTTTGCCGACACCGTGGCACCCCAACTCTCGCCCGGTGACATTGTTG

TCGCTCTTGGCCATACGCCTGTTGGCAGTATCTTCGACCTGAAGGTTGGTAACACCAAGCACACTCTCCAAGCCATCGAGACCAGGGTCCTTGCTGGG

TCCAAAATGACCGTGGCGCGCGTCGTCGATCCGACCCCCCACGCCCCCACCCGCACCCGTGCCCATCCCCCTCCCACCGAAGGTTTTGGAGAACGGTCC

AAACGCTTGGGGGGATGAAGACCGTTTGAATAAAAAGAGGAGGCGCAGGATGGAAGCCTCGGCATCTATGTTATGGGCGGGAAAAAGTACCAGAAAT

TTTGGGACAAGAATTCCGGTGACGTGTTTTATGAGGAGGTCCATAACAACACAGATGAGTGGGAGTGCCTCAGAGTTGGCGACCCTGCCGACTTTGAC

CCTGAGAAGGGAACTTTGTGTGGGCATGTCACCATTGAAGATAGGGCTTACCATGTTTACACCCTCCCCATCTGGTAAGAAATTCCTAGTCCCCGTCAA

CCCAGAGAACGGAAGAGTTCAATGGGAGGCTGCAAAGCTTTCCGTTGAGCAGGCCCTTGGTATGATGAACGTCGACGGCGAGCTGACTGCCAAGGAAC

TGGAGAAACTGAAAAGAATAATTGACAAACTCCAGGGCCTGACTAAGGAGCAGTGTTTAAACTGCTAGCCGCTAGCGGCTTGACCCGCTGTGGTCGCG

GCGGCTTGGTTGTTACTGAGACAGCGGTAAAAATAGTCAAATTTCACAAGCGGACCTTCACCTTGGGACCTGTAAATTTAAAAGTGGCTAGTGAGGTT

GAGCTAAAAGACGCGGTCGAGCACAACCAACACCCGGTTGCTAGACCAGTCGATGGTGGTGTTGTGCTCCTGCGTTCCGCGGTTCCCTCGCTTATAGA

TGTCCTGATCTCCGGTGCTGATGCATCTCCCAAATTACTTGCCCATCACGGACCGGGCAACACCGGGATCGATGGCACGCTCTGGGATTTTGAGTCCG

AAGCCACTAAAGAGGAAGTCGCACTCAGCGCGCAAATAATACAGGCTTGTGACATTAGGCGCGGCGACGCACCTGAAATTGGTCTCCCTTACAAGCTG

TACCCTATTAGGGGCAACCCTGAGCGGGTAAAGGGAGTTTTACGGAATACAAGGTTTGGAGACATACCTTATAAAACCCCAGTGACACTGGAAGCCC

AGTGCACGCGGCTGCCTGCCTCACGCCTAACACCACTCCGGTGACTGACGGGCGCTCCGTCTTGGCCACGACCATGCCCTCCGGGTTTGAGTTGTATG

TACCGACCATACCAGCGTCTGTCCTTGACTATCTTGATTCTAGGCCCGACTGCCCTAAACAGTTGACGGAGCACGGCTGTGAAGATGCCGCACTGAAA

GACCTCTCCAAGTATGACTTGTCCACCCAAGGCTTTGTTTTGCCTGGAGTTCTTCGCCTTGTGCGGAAGTACCTGTTTGCCCATGTGGGCAAATGCCC

GCCCGTTCATCGGCCTTCCACTTACCCTGCCAAGAATTCTATGGCTGGAATAAATGGGAACAGGTTTCCAACCAAGGACATCCAGAGCGTCCCTGAAA

TCGACGTTCTGTGCGCACAGGCTGTGCGAGAAAATTGGCAAACTGTTACCCCCTTGTACCCTCAAGAAACAGTATTGTGGGAAGAAGAAGACTAGGACC

ATACTCGGCA CC AATAACTTTATAGCG CT GGCTCACCGTGCAGCGTTGAGTGGTGTCACCCAGGGCTTCA T GAAAAAGGCGTTTAAC T CGCCTATCGC

CCTCGGGAAAAACAAATTTAAGGAGCTACAGACTCCGGTCCTAGGCAGGTGCCTTGAAGCTGATCTTGCATCCTGCGATCGATCTACACCTGCAATTG

TCCGCTGGTTTGCCGCCAATCTTCTTATGAGCTTGCCTGTGCTGAAGAGCATCTACCGTCGTATGTGCTGAACTGCTGTCACGACTTACTGGTCACG

CAGTCCGGTGCAGTGACTAAGAGAGGCGGCCTGTCGTCTGGCGACCCGATAACCTCTGTGTCTAATACCATTTTACAGCTTGGTGATCTATGCACAGCA

CATGGTGCTTAGTTGCTTCAAAAGTGGTCACCCCCATGGCCTTCTGTTCTTACAAGACCAGCTAAAGTTTGAGGACATGCTCAAAGGTTCAACCCTAA

TCGTCTATTCGGACGACCTCGTGCTGTATGCCGAGTCTCCCCACCATGCCAAACTACCATTGGTGGGTTGAACATCTGAATTTGATGCTGGGGTTTCAG

ACGGACCCAAAGAAAACAGCCATAACAGACTCGCCATCATTTCTAGGCTGTAGAATAATAAATGGGCGCCAGCTAGTCCCAACCGTGACAGGATTCT

CGCGGCCCTCGCCTACCACATGAAGGCGAGTAATGTTTCTGAATACTACGCCTCAGCGGCTGCAATACTCATGGACAGCTGTGCTTGTTTGGAGTATG

ATCCTGAATGGTTTGCAGAACTTGTAGTTGGGATAGCGCAGTGCGCCCGCAAAGACGGCTACAGCTTTCCCGGCACGCCGTTCTTCATGTCCATGTGG

GAAAAACTCAGGTCCAATTATGAGGGGAAGAAGTCGAGAGTGTGCGGGTACTGCGGGGCCCCGGCCCCGTACGCTACTGCCTGTGGCCTCGACGTCTG

CATTTACCACACCCACTTCCACCAGCATTGTCCAGTCACAATCTGGTGTGGCCATCCAGCGGGTTCTGGTTCTTGTAGTGAGTGCAAAATCCCCTGTAG

GGAAAGGCACAAGCCCTTTAGACGAGGTGCTGGAACAAGTCCCGTATAAGCCCCCACGGACCGTTATCATGCATGTGGAGCAGGGACTTACCCCCCTT

GACCCAGGTAGATACCAGACTCGCCGCGGATTAGTCTCTGTCAGGCGTGGAATCAGGGGAAATGAAGTTGAACTACCAGACGGTGATTATGCTAGTAC

CGCCTTGCTCCCTACCTGTAAAGAGATCAACATGGTCGCTGTCGCTTCTAATGTATTGCGCAGCAGGTTCATCGGTCCACCCGGTGCTGGGAAAA

CATACTGGCTCCTTCAACAGGTTCAGGATGGTGATGTTATTTACACCAACTCACCAGACCATGCTTGACATGATTAGAGCTTTGGGGACGTGCCGG

TTCAACGTTCCGGCAGGCACAACGCTGCAATTCCCCGTCCCCTCCCGTACCGGTCCGTGGGTTCGCATTCTAGCCGGCGGTTGGTGTCCTGGCAAGAA

TTCCTTCCTGGATGAAGCAGCATATTGCAATCACCTTGATGTTTTGAGGCTTCTTAGCAAAACTACCCTCACCTGTCTAGGAGACTTCAACAACTCC

ACCCAGTGGGTTTTGATTCTCATTGCTATGTTTTTTGACATCATGCCTCAAACTCAACTGAAGACCATCTGGAGGTTTGGACAGAATATCTGTGATGCC

ATCCAGCCAGATTACAGGGACAAACTCATGTCCATGGTCAACACAACCCGTGTGACCTACGTGGAAAAACCTATCAGGTATGGGCAAGTCCTCACCCC

CTACCACAGAGATCGAGAGGACGACGCCATCACTATTGACTCCAGTCAAGGCGCCACATTCGATGTGGTTACACTGCATTTGCCCACTAAAGATTCAC

TCAACAGGCAAAGAGCCCTTGTTGCTATCACCAGGGCCAGACATGCTATCTTTGTGTATGACCCACACAGGCAGCTGCAGAGCTTGTTTGATCTTCCT

GAAAAAGGCACACCCGTTAACCTCGCAGTGCACCGCGACGGGCAGCTGATCGTGCTGGATAGAAATAACAAAGAATGCACGGTTGCTCAAAGCTCTAGG

CAACGGGGATAAATTTAGGGCCACAGACAAGCGTGTTGTAGATTCTCTCCGCGCTATTTGTGCTGACCTAGAAGGGTCGAGCTCTCCGCTCCCCAAGG

TCGCACACAACTTGGGATTTTATTTCTCACCTGATTTAACACAGTTTGCCAAACTCCCAGCAGAACTTGCACCTCACTGGCCTGTGGTGACAACCCAG

AACAATGAAAATTGGCCAGATCGGCTGGTTGCCAGCCTTCGTCCTATCCATGAGTATAGCCGCGCGTGCATCGGTGCCGGCTATATGGTTGGCCCCTC

GGTGTTTCTAGGCACCCCCGGGGTCGTGTCATACTATCTCACAAAATTTGTTAAGGGCGAGGCTCAAGTGCTTCCGAAGACGGTCTTCAGCACCGGCC

GAATTGAGGTAGATTGCCGGGAATATCTTGACGATCGGGAGCGAGAAGTTGCTGCGTCCCTCCCACATGCCTTCATTGGTGACGTCAAAGGCACTACC

GTTGGAGGATGTCATCATGTCACCTCCAGATACCTCCCGCGCTTCCTCCCCAAGGAATCAGTTGCTGTGGTCGGGGTTTCAAGTCCCGGAAAAGCCGC

GAAAGCATTGTGCACACTGACAGATGTGTACCTCCCCAGACCTTGAAGCCTATCTCACCCGGAGACCCAGTCCAAGTGCTGGAAAATGATGTTGGACT

TCAAGGAAGTTCGACTAATGGTCTGGAAAGACAAAACAGCCTATTTCCAACTTGAAGGACGCTATTTCACCTGGTATCAGCTTGCTAGCTATGCCTCG

TACATCCGTGTTCCTGTCAACTCTACTGTGTACTTGGACCCCTGCATGGGCCCCGCCCTTTGCAACAGGAGAGTCGTTGGGTCCACTCACTGGGGGGC

TGACCTCGCAGTCACCCCCTTATGATTACGGCGCTAAAATTATCCTGTCTAGCGCGTACCATGGTGAAATGCCCCCCGGATACAAAATTCTGGCGTGCG

CGGAATTCTCGTTGGATGACCCAGTCAAGTACAAACATACTTGGGGGTTTGAATCGGATACAGCGTATCTGTATGAGTTCACCGGAAACGGTGAGGAT

TGGGAGGATTACAATGATGCGTTTCGTGCGCGCCAGGAAGGGAAAATTTATAAGGCCACTGCCACCAGCATGAAGTTTTATTTTCCCCCGGGCCCTGT

CATTGAACCAACTTTAGGCCTGAATTGATTAATTAATTTAAAAGGCGCGCCAATGAAATGGGGTCCATGCAAAAGCCTTTTTGACAAAATTGGTCAACT

TTTTGTGGATGCTTTCACGGAATTCTTGGTGTCCATTGTTGATATCATCATATTTTGGCCATTTTGTTTGGCTTCACCGTCGCCGGTTGGCTGGTGG

TCTTTTGCATCAGATTGGTTTGCTCCGCGATACTCCGTGCACGCCCTGCCTTTCACTCTGAGCAGTTACAGAAGATCCTATGAGGCCTTTCTTTCCCT

GTGTCAGGTGGACATTCCCACCTGGGGAATCAAACATCCTCTGGGGGTGCTTTGGCACCATAAGGTGTCAACCCTGATTGATGAAATGGTGTCGCGTC

GAATGTACCGCATCATGGAAAAAGCAGGACAGGCTGCCTGGAAACAGGTGGTGAGCGAGGCCACGCTGTCTCGCATTAGTAGTTTGGATGTGGTGGCT

CATTTTCAACATCTTGCCGCCGTTGAAGCCGAGACCTGTAAATATTTGGCCTCTCGGCTACCCATGCTACACAACCTGCGCATGACGGGGTCAAATGT

AACCATAGTATATAATGGTACTTTGAATCAGGTGTTTGCCATTTTCCCGACCCCTGGTTCCCGGCCAAAGCTTCATGATTTTCAGCAATGGCTGATAG

CTGTGCATTCGTCCATATTTTTCCTCTGTTGCAGCTTCTTGTACTCTGTTTGTTGTACTGTGGTTGCGGGTCCCAATGCTACGTACTGTTTTTGGTTTC

CGCTGGTTAGGGGCAATTTTTCCTTCGAGCTCTTGGTGAATTACACGGTGTGCCCCACCTTGCCTCACCCGGCAAGCAGCCGCACAGCGCTACGAACCT

GGCAAGGCTTTTTGGTGCAGAATTGGGTACGATCGATGTGAGGAGGACGATCACGACGAGCTAGGGTTCGTGATACCGTCTGGCCTCTCCAGCGAAGG

CCACTTGACTAGTGT TACGCCTGGCTGGCG TT CCTGTCCTTCAGCTACACGGCCCAGTTCCATCCCGAGATAT TT GGGATAGGGAATGTG AG TCAAG

TTTATGTTGACATCAAGCACCAATTCATCTGCGCTGTTCACGACGGGGATAACGCCACCTTGCCTCGCCATGACAATATTTCAGCCGTATTTCAGACC

TACTACCAACACCAGGTCGACGGCGGCAATTGGTTTCACCTGGAATGGCTGCGTCCTTTCTTTTCCTCTTGGTTGGTTTTAAATGTTTCGTGGTTTCT

CAGGCGTTCGCCTGCAAGCCATGTTTCAGTTCGAGTCTTTCGGACATCAAAACCAACACCACCGCAGCATCAGACTTCGTTGTCCTCCAGGACATCAG

CTGCCTTAGGCATGGCGACTCGTCCTCTCCGACGATTCGCAAAATTTCTCAGTGCCGCACGGCGATAGGGACGCCCGTGTACATCACCATCACTGCCA

ATGTCACAGATGAAAATTATCTACATTCTTCTGATCTCCTCATGCTTTCTTCTTGCCTTTTCTATGCTTCCGAGATGAGTGAAAAGGGATTCAAAGTG

GTGTTTGGCAATGTGTCAGGCACTGTGGCTGTGTGCGTCAACTTTACCAGCTACGTCCAACATGTCAAGGAGTTTACCCAACGCTCCTTAGTGGTCGA

TCATGTGCGACTGCTTCATTTCATGACACCTGAGACCATGAGGTGGGCAACCGTTTTTAGCCTGTCTTTTTGCCATCCTACTGGCAATTTGAATGTTCA

AGTATGTTGGGGAAGTGCTTGACCGCGTGCTGTTGCTCGCGATTGCTTTTTTTGTGGTGTATCGTGCCGTTCTATCTTGCTGTGCTCGTCAACGCCAG

CAACAACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGTTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGG

AGACTTTTGTCATTCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGGCACTCACCAGCCATTTCCTTGACACAGTTGGTTTGGCCACTGTGTCT

ACCGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAA

CTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTGTGGAGA

AAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCTTTAACCAGAGTTTCAGCGGAA

CGATGGGGTCGTCTCTAGACGACTTCTGCAATGATAGCACAGTCCACAGAAGGTGCTTTTGGCGTTTTCCATTACCTACACGCCAGTGATGATATAT

GCTCTAAAGGTAAGTCGCGGCCGACTGCTAGGGCTTCTGCACCTTTTGATCTTTCTGAATTGTGCTTTTACCTTCGGGTACATGACATTCGTGCACTT

TGAGAGCACAAATAGGGTCGCGCTCACTATGGGAGCAGTAGTTGCACTTCTTTGGGGAGTGTACTCAGCCATAGAAACCTGGAAATTCATCACCTCCA

GATGCCGTTTGTGCTTGCTAGGCCGCAAGTACATTCTGGCCCCTGCCCACCACGTCGAAAGTGCCGCGGGCTTTCATCCGATTGCGGCAAATGATAAC

CACGCATTTGTCGTCCGGCGTCCCGGTCCCACTACGGTCAACGGCACATTGGTGCCCGGGTTGAAAAGCCTCGTGTTGGGTGGCAGAAAAGCTGTTAA

GCAGGGAGTGGTAAACCTTGTTAAATATGCCAAATAACAACGGCAAGCAGCAAAAGAAAAAGAAGGGGAATGGCCAGCCAGTCAATCAGCTGTGCCAA

ATGCTGGGTAAGATCATCGCCCAACAAAACCAGTCCAGAGGCAAGGGACCGGGGAAGAAAAATAGGAAGAAAAACCCGGAGAAGCCCCATTTCCCTCT

AGCGACTGAAGATGACGTCAGGCATCACTTTACCCCTAGTGAGCGGCAATTGTGTCTGTCGTCGATCCAGACTGCCTTCAATCAGGGCGCTGGAACTT

GTGCCCTGTCAGATTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCGACGCAACATACTGTGCGTCTGATCCGCGCCACAGCATCACCCTCA

GCATGATGGGCTGGCATTCTTTGGCACCTCAGTGTTAGAATTGGGAGAATGTGTGGTGAATGGCACTGATTGACACTGTGCCTCTAAGTCACCTATTC

AATTAGGGCGACCGTGTGGGGGTAAAAGTTTAATTGGCGAGAACCATGCGGCCGTAATTAAAAAAAAAAAAA

Claims (4)

1, porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain, after utilizing reverse genetic manipulation and gene recombination technology all nucleotide sequence Delete Alls with the latter half of the GP2 gene of the classical malicious PTK strain of porcine reproductive and respiratory syndrome virus (PRRSV) and GP3～GP5 gene, M protein gene, N protein gene and intervening sequence thereof and 3 ' non-coding region, replace to the corresponding gene of the high pig blue-ear disease variation strain of causing a disease of China, reassemble into a chimeric recombinant vaccine strain (PC strain) that contains classical strain of porcine reproductive and respiratory syndrome and variant gene simultaneously.

2, porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain according to claim 1, it is characterized in that: the high corresponding gene that causes a disease pig blue-ear disease variation strain of described China is latter half, GP3 gene, GP4 gene, GP5 gene, M protein gene, N protein gene and the intervening sequence thereof of the Chinese highly pathogenic PRRS GP2 of variation strain gene and all nucleotide sequences of 3 ' non-coding region, and the purpose nucleotide sequence total length that inserts is 2421bp, the structural protein of the PRRSV that mainly encodes and 3 ' non-coding region.

3, the porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain according to claim 1 and 2 method of producing living vaccine, carry out according to the following steps:

(1) seedling going down to posterity and cultivating with cell: Marc145 or MA104 clone through EDTA-pancreatin cell dispersion liquid had digestive transfer culture, are continued to cultivate with cell growth medium, when forming individual layer, standby;

(2) breeding of cell seed culture of viruses: the cell bottle that PC strain virus liquid has been grown into good cell monolayer by 1/10 volume access, adsorb after 1 hour, add cell maintenance medium in the clone individual layer, continue to cultivate, after appearring in the 70-80% cell, pathology gathers in the crops, 2-3 rearmounted preservation below-15 ℃ of the venom freeze thawing of results takes a morsel and does the inspection of semifinished product;

(3) join seedling, packing and freeze-drying: with the virus-culturing fluid that is up to the standards, mix in a container by 1: 1 volume ratio, fully shake up, quantitatively packing with conventional stablizer; Every part contains the cell venom and is no less than 10 ^5.0TCID ₅₀, carry out lyophilize after the packing and get product.

4, the detection method of the transgenic microorganism of porcine reproductive and respiratory syndrome virus chimeric recombinant vaccine strain PC according to claim 1 and 2 strain adopts the RT-PCR method to detect.

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