阅读说明：本技术 一种针对ii类vii型流行ndv株dhn3的感染性重组克隆方法 (Infectious recombinant cloning method for II-type VII-type epidemic NDV strain DHN3 ) 是由 陈瑞爱 黄梅 王楠楠 李延鹏 刘定祥 叶俊贤 罗琼 杨小云 董楠 于 2019-09-20 设计创作，主要内容包括：本发明属于兽用生物制品技术领域,公开了一种针对II类VII型流行NDV株DHN3的感染性重组克隆方法,包括如下步骤：步骤1：构建辅助质粒,所述辅助质粒为三种,辅助质粒上的目标片段分别为NP基因、P基因、L基因；步骤2：构建DHN3全基因组表达载体；将人工重组得到的DHN3全基因组重组入载体质粒中得到全基因组表达载体；DHN3全基因组的序列如序列表SEQ ID NO 1；步骤3：将三种辅助质粒和DHN3全基因组表达载体共转染BHK-21细胞,得到含重组病毒rDHN3的病毒液；NP基因在序列表SEQ ID NO：1中位置为1-1591nt；P基因在序列表SEQ ID NO：1中位置为1925-3109nt；L基因在序列表SEQ ID NO：1中位置为8166-15192nt。该方法可以成功的克隆出于原病毒株完全相同、无突变的重组病毒。(The invention belongs to the technical field of biological products for livestock, and discloses an infectious recombinant cloning method for a II-class VII epidemic NDV strain DHN3, which comprises the following steps: step 1: constructing three types of helper plasmids, wherein target fragments on the helper plasmids are NP gene, P gene and L gene respectively; step 2: constructing a DHN3 whole genome expression vector; the DHN3 whole genome obtained by artificial recombination is recombined into a vector plasmid to obtain a whole genome expression vector; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1; and step 3: co-transfecting the three auxiliary plasmids and a DHN3 whole genome expression vector to a BHK-21 cell to obtain virus liquid containing recombinant virus rDNN 3; the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt. The method can successfully clone the recombinant virus which is completely the same as the original virus strain and has no mutation.)

1. An infectious recombinant cloning method for the circulating NDV strain DHN3 of type II VII, comprising the steps of:

step 1: constructing three types of helper plasmids, wherein target fragments on the helper plasmids are NP gene, P gene and L gene respectively;

step 2: constructing a DHN3 whole genome expression vector; the DHN3 whole genome obtained by artificial recombination is recombined into a vector plasmid to obtain a whole genome expression vector; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1;

and step 3: co-transfecting the three auxiliary plasmids and a DHN3 whole genome expression vector to a BHK-21 cell to obtain virus liquid containing recombinant virus rDNN 3;

the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt.

2. The infectious recombinant cloning method of the circulating type II VII NDV strain DHN3 according to claim 1, wherein: the vector involved in the helper plasmid in the step 1 is pXJ40 series plasmid or pcDNA 3; the vector plasmid in the step 2 is a pBR322 series plasmid or a pACYC and pBAD series plasmid.

3. The infectious recombinant cloning method of the circulating type II VII NDV strain DHN3 according to claim 1, wherein: the method comprises the following steps:

step 1: constructing helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L, wherein target fragments of the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L are NP gene, P gene and L gene respectively, and a vector is pXJ 40;

step 2: constructing a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome obtained by artificial recombination is recombined into pBR322 plasmid to obtain a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome is the whole genome of II-type VII-type newcastle disease virus;

and step 3: helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 are co-transfected into BHK-21 cells to obtain virus liquid containing recombinant virus rDNN 3.

4. The infectious recombinant cloning method of the circulating type II VII NDV strain DHN3 according to claim 3, wherein: the step 2 specifically comprises the following steps:

step 21: establishing a pBR322-Base vector; introducing a fragment capable of homologous recombination with the whole genome of DHN3 into a pBR322 plasmid; the fragment has homology arms corresponding to the 3 'end and the 5' end of the DHN3 whole genome;

step 22: constructing a transition vector; the transition vector is plasmid pBR322-PNP, plasmid pBR322-PDP and plasmid pBR322-LPD 3; the target fragment of the plasmid pBR322-PNP comprises NP, MINI and P genes; the target fragment of the plasmid pBR322-PDP comprises P, PD1 and PD2, PD3 genes; the target fragment of the plasmid pBR322-LPD3 comprises L1, L2, L3, L4 and PD3 genes;

step 23: constructing DHN3 whole genome DHN 3-A; carrying out enzyme digestion on the plasmid pBR322-PNP, the plasmid pBR322-PDP and the plasmid pBR322-LPD3 to obtain gene fragments PNP, LPD3 and PDP, and connecting the gene fragments PNP, LPD3 and PDP through T4 ligase to obtain DHN3 whole genome DHN 3-A;

step 24: constructing a plasmid fragment with a homology arm; carrying out PCR amplification by taking the pBR322-Base vector in the step 21 as a template to obtain a plasmid fragment with a homology arm;

step 25: constructing a whole genome expression vector pBR322-DHN 3; carrying out homologous recombination on the plasmid fragment with the homologous arm in the step 24 and the DHN3 whole genome DHN3-A in the step 23 to obtain a plasmid pBR322-DHN3 with the DHN3 whole genome DHN 3-A;

the MINI gene is shown in a sequence table SEQ ID NO: the 1 position is 1414-; the PD1 gene is shown in a sequence table SEQ ID NO: the 1-position is 2935-; the PD2 gene is shown in a sequence table SEQ ID NO: the 1 middle position is 4838 and 6454 nt; the PD3 gene is shown in a sequence table SEQ ID NO: the 1 position is 6261 and 8283 nt; the L1 gene is shown in a sequence table SEQ ID NO: the position in the 1 position is 8166 + 10709 nt; the L2 gene is shown in a sequence table SEQ ID NO: the 1-position is 10174 and 12299 nt; the L3 gene is shown in a sequence table SEQ ID NO: the 1-position is 12238-; the L4 gene is shown in a sequence table SEQ ID NO: position 14214- > 15192nt in 1.

5. The infectious recombinant cloning method of the type II, type VII epidemic NDV strain DHN3 according to claim 4, wherein said step 3 is specifically:

co-transfecting BHK-21 cells with plasmids capable of expressing T7RNA polymerase, helper plasmids pXJ40-NP, pXJ40-P, pXJ40-L and a whole genome expression vector pBR322-DHN 3; obtain virus liquid containing recombinant virus rDNN 3.

6. The infectious recombinant cloning method of the type II, type VII epidemic NDV strain DHN3 according to claim 4, wherein said step 21 comprises:

introducing 1T 7 promoter, 1T 7 terminator and 1 HDV Ribozyme into pBR322 plasmid; partial base HC1 introduced to the 3' end of DHN3 downstream of the T7 promoter; the base HC2 in the 5' terminal part of DHN3 was introduced upstream of HDV Ribozyme; wherein, the position sequence of the base HC1 in the DHN3 whole genome sequence is 15192-15159 nt; the base HC2 has a position sequence of 141-1nt in the whole genome sequence of DHN 3.

7. The infectious recombinant cloning method of the type II, type VII epidemic NDV strain DHN3 according to claim 4, wherein said step 22 comprises:

step 221: preparation of the pBR322 plasmid fragment: carrying out double enzyme digestion on pBR322 plasmid by Hind3 and Nhe1, and then recovering for later use by gel;

step 222: preparing a target fragment; the target fragment is NP, MINI, P, PD1, PD2, PD3, L1, L2, L3 and L4 genes;

step 223: the pBR322 plasmid fragment and the corresponding target fragment are linked by recombinase to obtain the corresponding plasmids pBR322-PNP, pBR322-PDP and pBR322-LPD 3.

8. The method of infectious recombinant cloning of the circulating type II VII NDV strain DHN3 according to claim 4, wherein said step 23 is:

plasmid pBR322-PDP and plasmid pBR322-LPD3 are digested separately with BtgZ1 to recover gene fragment LPD and PDP; the vector pBR322-PNP is subjected to double enzyme digestion by BtgZ1 and Hind3 to recover a gene fragment PNP;

the gene fragment LPD, the gene fragment PDP and the gene fragment PNP are connected in vitro through T4 ligase to obtain DHN3 whole genome DHN 3-A.

Technical Field

The invention relates to the technical field of veterinary biological products, in particular to an infectious recombinant cloning method aiming at a II-type VII epidemic NDV strain DHN 3.

Background

Newcastle disease is a highly contagious and lethal disease caused by Newcastle Disease Virus (NDV) that mainly attacks chickens, turkeys, wild birds and ornamental birds, commonly known as fowl plague. Belongs to highly contact, acute and severe infectious diseases. Humans occasionally become infected, manifesting as conjunctivitis. The world health Organization (OIE) lists the infectious diseases as the infectious diseases which need to be reported, and the department of agriculture in China also lists the infectious diseases as a type of animal epidemic diseases which need to be reported. Due to the rapid propagation speed and the 100% incidence and fatality rate, once the spread seriously harms the poultry industry, immeasurable loss is caused.

The prevention of newcastle disease is a key measure in addition to rational feeding. In addition to establishing a well-established and correct immunization program, the provision of an excellent broad-spectrum, highly effective and inexpensive vaccine is fundamental to the prevention of the disease.

Newcastle disease vaccines are divided into inactivated and live vaccines. The live seedlings mainly include line I, line II (B1 strain), line III (F strain) and line IV (Lasota strain). Wherein the IV-series live vaccine (Lasota strain) is an excellent low-toxicity vaccine widely applied at home and abroad at present, has higher toxicity and immunity than the II-series vaccine, and has good safety. With the application of cloning technology, a new generation of cloned seedlings is gradually coming into the market. The new generation of attenuated live vaccine favored by the market at present mainly comprises a Newcastle disease clone live vaccine C/30 developed by the Netherlands and an IV line optimized clone vaccine developed by the American general knowledge. In addition, the live vaccine (I line clone vaccine) of the moderate-virulence clone strain in the Newcastle disease also has the advantages of mild virulence and strong safety similar to the common I line vaccine.

According to epidemiological investigations, the epidemic genotype of NDV varies with time and geographical environment. In the last 20-50 years, the fluid I-IV type was predominant, and in the 70 th year, the V type was first found to be predominant in south America and middle America so as to be in Europe. Type VI appeared in the 80 s and prevailed in the middle east, asia and europe. Type VII began to spread in 85 years and, with type VIII spreading in many countries of the world and in the 90 s, resulted in a pandemic in asia, africa and the middle east. The V-VIII types are all strong toxins. Types IX and X have been limited to local sporadic states. Because vaccines against type IV are widely used, type IV NDV has been well controlled. However, vaccines against other genotypes are still lacking and tend to be popular. Therefore, the reverse genetic cloning technology is utilized to rapidly develop the attenuated vaccine which is high-efficiency, broad-spectrum, safe and even multivalent aiming at the real-time epidemic strains. In addition, quarantine is made more difficult by the widespread use of vaccines. Because it is impossible to discriminate whether the infected chicken carries a vaccine strain or a wild strain. An artificial label can be introduced in a recombination mode, so that the artificial label can be effectively identified with wild viruses.

Newcastle Disease Virus (NDV) belongs to the order Mononegales, Paramyxoviridae. The virus has a double lipid layer envelope lined with a layer of M protein. The outer membrane is coated with fiber-like glycoproteins (HN and F) to give a panicle-like appearance. The capsule contains a long helical nucleocapsid composed of a capsid protein and a minus-strand RNA. Newcastle disease virus has 6 groups of genes encoding 6 viral proteins, i.e., hemagglutinin and neuraminidase active (HN) glycoprotein, fusion functional (F) glycoprotein, non-glycosylated inner membrane protein (M), Nucleocapsid Protein (NP), phosphoprotein (P) and high molecular weight protein (L), respectively. The total length of the NDV gene is 15186nt to 15198 nt.

It has been shown that the cloning of NDV cDNA by artificial mutation, replacement, or insertion of foreign sequences does not prevent the replication, assembly and release of the virus. cDNA clones of NDV have been used for basic research and vaccine development. The cDNA clone of the NDV can be used as a vector to express antigen proteins of other pathogenic bacteria so as to obtain the multivalent vaccine for resisting various pathogenic bacteria. The weak toxicity of the Latasa strain is changed into strong toxicity by base mutation of F gene cleavage points by Peeters and the like in 1999 (ref 1); in 2004 Huang et al exchanged the HN genes of the virulent and attenuated strains to obtain a new strain with different virulence (ref 2). In 2002, Mebastson et al successfully obtained a hybrid virus (ref3) which resists both NDV and hepatitis virus by replacing the NP protein dominant epitope of NDV with the S2 glycoprotein epitope gene of hepatitis virus; in 2006, Man et al inserted the HA gene of H7 avian influenza virus between the P-M genes of NDV B1 strain and succeeded in obtaining a hybrid virus (ref4) that is resistant to both NDV and H7 avian influenza viruses. Recently Abzeid et al successfully assembled the currently circulating IBV S protein of Egypt into recombinant NDV, resulting in a hybrid virus that is immune protected against both the original NDV and the corresponding IBV. (ref5) Chinese scholars also use LaSota strain as carrier to obtain multiple bivalent hybrid viruses. Such as the hybrid virus resistant to both NDV and IBDV5 (ref 6); hybrid virus resistant to both NDV and H5N1 (ref 7); hybrid virus resistant to both NDV and Mycoplasma gallisepticum TM1 virus (ref 8). Because NDV has only one serotype, its genetic properties are relatively stable. Although infectious cDNA clones of NDV have been established internationally, they are limited to the Latasa strain and to the basic and clinical research level, which may be related to the technical capabilities of the vaccine production units in China.

Therefore, the technical problem to be solved by the present application is: how to develop infectious recombinant clonal viruses aiming at the II-type VII epidemic NDV strain DHN 3.

Disclosure of Invention

The invention aims to provide an infectious recombinant cloning method aiming at a II-type VII epidemic NDV strain DHN3, which can successfully clone recombinant viruses which are completely the same as original virus strains and have no mutation.

In order to achieve the purpose, the invention provides the technical scheme that: an infectious recombinant cloning method for a type II VII epidemic NDV strain DHN3, comprising the following steps:

step 1: constructing three types of helper plasmids, wherein target fragments on the helper plasmids are NP gene, P gene and L gene respectively;

step 2: constructing a DHN3 whole genome expression vector; the DHN3 whole genome obtained by artificial recombination is recombined into a vector plasmid to obtain a whole genome expression vector; the whole genome sequence of DHN3 is shown in a sequence table SEQ ID NO 1;

and step 3: co-transfecting the three auxiliary plasmids and a DHN3 whole genome expression vector to a BHK-21 cell to obtain virus liquid containing recombinant virus rDNN 3;

the NP gene is shown in a sequence table SEQ ID NO: the 1 middle position is 1-1591 nt; the P gene is shown in a sequence table SEQ ID NO: the position in the 1 is 1925 and 3109 nt; the L gene is shown in a sequence table SEQ ID NO: the 1 position is 8166 + 15192 nt.

In the above infectious recombinant cloning method against the II type VII epidemic NDV strain DHN3, the vector involved in the helper plasmid in the step 1 is pXJ40 series plasmid or pcDNA 3; the vector plasmid in the step 2 is a pBR322 series plasmid or a pACYC and pBAD series plasmid.

In the above infectious recombinant cloning method for the circulating NDV strain type II VII DHN3, the following steps are included:

step 1: constructing helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L, wherein target fragments of the helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L are NP gene, P gene and L gene respectively, and a vector is pXJ 40;

step 2: constructing a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome obtained by artificial recombination is recombined into pBR322 plasmid to obtain a whole genome expression vector pBR322-DHN 3; the DHN3 whole genome is the whole genome of II-type VII-type newcastle disease virus;

and step 3: helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 are co-transfected into BHK-21 cells to obtain virus liquid containing recombinant virus rDNN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 2 is specifically:

step 21: establishing a pBR322-Base vector; introducing a fragment capable of homologous recombination with the whole genome of DHN3 into a pBR322 plasmid; the fragment has homology arms corresponding to the 3 'end and the 5' end of the DHN3 whole genome;

step 22: constructing a transition vector; the transition vector is plasmid pBR322-PNP, plasmid pBR322-PDP and plasmid pBR322-LPD 3; the target fragment of the plasmid pBR322-PNP comprises NP, MINI and P genes; the target fragment of the plasmid pBR322-PDP comprises P, PD1 and PD2, PD3 genes; the target fragment of the plasmid pBR322-LPD3 comprises L1, L2, L3, L4 and PD3 genes;

step 23: constructing DHN3 whole genome DHN 3-A; carrying out enzyme digestion on the plasmid pBR322-PNP, the plasmid pBR322-PDP and the plasmid pBR322-LPD3 to obtain gene fragments PNP, LPD3 and PDP, and connecting the gene fragments PNP, LPD3 and PDP through T4 ligase to obtain DHN3 whole genome DHN 3-A;

step 24: constructing a plasmid fragment with a homology arm; carrying out PCR amplification by taking the pBR322-Base vector in the step 21 as a template to obtain a plasmid fragment with a homology arm;

step 25: constructing a whole genome expression vector pBR322-DHN 3; carrying out homologous recombination on the plasmid fragment with the homologous arm in the step 24 and the DHN3 whole genome DHN3-A in the step 23 to obtain a plasmid pBR322-DHN3 with the DHN3 whole genome DHN 3-A;

the MINI gene is shown in a sequence table SEQ ID NO: the 1 position is 1414-; the PD1 gene is shown in a sequence table SEQ ID NO: the 1-position is 2935-; the PD2 gene is shown in a sequence table SEQ ID NO: the 1 middle position is 4838 and 6454 nt; the PD3 gene is shown in a sequence table SEQ ID NO: the 1 position is 6261 and 8283 nt; the L1 gene is shown in a sequence table SEQ ID NO: the position in the 1 position is 8166 + 10709 nt; the L2 gene is shown in a sequence table SEQ ID NO: the 1-position is 10174 and 12299 nt; the L3 gene is shown in a sequence table SEQ ID NO: the 1-position is 12238-; the L4 gene is shown in a sequence table SEQ ID NO: position 14214 + 15192nt in 1.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 3 is specifically:

co-transfecting BHK-21 cells with plasmids capable of expressing T7RNA polymerase, helper plasmids pXJ40-NP, pXJ40-P, pXJ40-L and a whole genome expression vector pBR322-DHN 3; obtain virus liquid containing recombinant virus rDNN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 21 is specifically:

introducing 1T 7 promoter, 1T 7 terminator and 1 HDV Ribozyme into pBR322 plasmid; partial base HC1 introduced to the 3' end of DHN3 downstream of the T7 promoter; the base HC2 in the 5' terminal part of DHN3 was introduced upstream of HDV Ribozyme; wherein, the position sequence of the base HC1 in the DHN3 whole genome sequence is 15192-15159 nt; the base HC2 has a position sequence of 141-1nt in the whole genome sequence of DHN 3.

In the above infectious recombinant cloning method for the type II VII epidemic NDV strain DHN3, the step 22 is specifically:

step 221: preparation of the pBR322 plasmid fragment: carrying out double enzyme digestion on pBR322 plasmid by Hind3 and Nhe1, and then recovering for later use by gel;

step 222: preparing a target fragment; the target fragment is NP, MINI, P, PD1, PD2, PD3, L1, L2, L3 and L4 genes;

step 223: the pBR322 plasmid fragment and the corresponding target fragment are linked by recombinase to obtain the corresponding plasmids pBR322-PNP, pBR322-PDP and pBR322-LPD 3.

In the above infectious recombinant cloning method for the circulating NDV strain type II VII DHN3, the step 23 is:

plasmid pBR322-PDP and plasmid pBR322-LPD3 are digested separately with BtgZ1 to recover gene fragment LPD and PDP; the vector pBR322-PNP is subjected to double enzyme digestion by BtgZ1 and Hind3 to recover a gene fragment PNP;

the gene fragment LPD, the gene fragment PDP and the gene fragment PNP are connected in vitro through T4 ligase to obtain DHN3 whole genome DHN 3-A.

The invention has the beneficial effects that:

the method of the invention can successfully clone the recombinant virus which is completely the same as the original virus strain and has no mutation.

The total genome of DHN3 is 15192nt, and if the full-length cDNA is obtained by RT-PCR at one time, the difficulty is that the general commercially available DNA polymerase can hardly synthesize DNA fragments larger than 10k, and the random mutation can be easily introduced in the PCR process. The DHN3 whole genome was therefore divided into 3 fragments, which were cloned separately into the pBR322 vector. The size of the fragment was determined based on the BtgZ1 cleavage site it holds to facilitate subsequent in vitro DNA fragment ligation.

The invention adopts helper plasmids pXJ40-NP, pXJ40-P and pXJ40-L and a whole genome expression vector pBR322-DHN3 to prepare virus liquid, and the main reasons are as follows: because the whole genome expression vector pBR322-DHN3 transfected into cells is limited, enough structural and functional proteins cannot be synthesized in cells in a short period of time; the components necessary for viral replication and assembly can be provided in a short period of time by co-transfecting helper genes.

Drawings

FIG. 1 is a diagram showing the alignment of the sequencing result of the first embodiment of the present invention with all published NDV sequences.

FIG. 2 is a micrograph showing that NDV-infected BHK-21 cells undergo significant cell fusion according to the first embodiment of the present invention;

FIG. 3 is a diagram showing the relationship between the L gene and sequencing plasmids pMD19-L1, pMD19-L2, pMD19-L3 and pMD19-L4 in the first embodiment of the present invention;

FIG. 4 is an electrophoretogram of the PCR product of helper plasmid pXJ40-L according to a first embodiment of the present invention;

FIG. 5 is an electrophoretogram of the PCR product of helper plasmid pXJ40-L according to a first embodiment of the present invention;

FIG. 6 is a restriction electrophoresis diagram of helper plasmid pXJ40-L according to the first embodiment of the present invention;

FIG. 7 is a plasmid schematic diagram of plasmid pXJ40-L, which is a first embodiment of the present invention;

FIG. 8 is a plasmid schematic diagram of plasmid pXJ40-P, which is a first embodiment of the present invention;

FIG. 9 is a plasmid schematic diagram of the plasmid pXJ40-NP according to the first embodiment of the present invention;

FIG. 10 is a plasmid schematic diagram of a pBR322-Base vector according to the first embodiment of the present invention;

FIG. 11 is a plasmid schematic diagram of plasmid pBR322-PNP according to the first embodiment of the present invention;

FIG. 12 is a plasmid diagram of plasmid pBR322-PDP according to the first embodiment of the present invention;

FIG. 13 is a plasmid schematic diagram of plasmid pBR322-LPD3 according to the first embodiment of the present invention;

FIG. 14 is a schematic structural diagram of a full-length DHN3-A according to a first embodiment of the present invention;

FIG. 15 is an electrophoretogram of positive colonies A1-A3 of the first example of the present invention;

FIG. 16 is a restriction enzyme electrophoresis diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 17 is a plasmid schematic diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 18 is a plasmid schematic diagram of the whole genome expression vector pBR322-DHN3 according to the first embodiment of the present invention;

FIG. 19 is a graph showing the growth characteristics of DHN3 virus, rDNN virus, and a blank control according to a first embodiment of the present invention;

FIG. 20 is a colony electrophoretogram of PCR of pXJ40DE3 plasmid in accordance with the first embodiment of the present invention;

FIG. 21 is a plasmid schematic diagram of a pBR322 plasmid fragment according to the first embodiment of the present invention;

FIG. 22 is a plasmid schematic of plasmid pXJ40 according to the first embodiment of the present invention;

FIG. 23 is a plasmid schematic of plasmid pXJ40-DE3, which is one embodiment of the present invention.

Detailed Description

The invention will be described with reference to specific embodiments: it is to be understood that these specific embodiments are merely illustrative of the invention and are not to be construed as limiting the invention. While those skilled in the art can fully appreciate the improvements to the embodiments and features of the present invention that can be made without departing from the scope of the present invention, such improvements and modifications are intended to be included within the scope of the present invention.